TW201412329A - Yeast-based vaccine for inducing an immune response - Google Patents

Abstract

The invention provided herein relates to vaccines that can be tailored to achieve a desired immune response. Some compositions provided herein are used for preferentially eliciting a humoral immune response while other compositions are useful for preferentially eliciting a cell-mediated response. Combinations of vaccine compositions are also useful for eliciting both types of responses and/or for modulating the type of immune response elicited. The invention also provides methods for eliciting an immune response in an individual by administering the compositions disclosed herein. These immune responses are useful for protecting an individual from various types of diseases, infections, and undesirable conditions.

Description

Yeast-based vaccine for inducing an immune response

The present invention is generally directed to yeast-based vaccines that can induce various types of protective and therapeutic immune responses, including cell-mediated and/or humoral immune responses. Compositions comprising such yeast-based vaccines and methods of using such vaccines, including methods of using such vaccines in combination with other types of vaccines, are disclosed herein. Various compositions and methods are also disclosed for vaccinating animals to prevent influenza infection and to treat or prevent influenza infection in animals.

Vaccines are one of the most cost-effective methods that can be used by the health care industry to prevent and treat diseases. However, there is an urgent need to develop safe and effective vaccines and adjuvants for various diseases, including diseases caused by or associated with pathogen infections, cancer, genetic defects, and other conditions of the immune system. Regarding the disclosure of vaccines, for example, Rabinovich et al, Science 265, 1401-1404 (1994), indicates that a safe and thermally stable vaccine is still needed, which can be administered orally and requires only a few times, preferably in the early stages of life. . Combination vaccines that protect individuals from more than one disease and vaccines that do not require adjuvants and that induce cell-mediated immunity, humoral immunity, and mucosal immunity are also preferred. To date, there are very few vaccines (if any) that meet all of these criteria.

Dead or attenuated pathogens are commonly used in conventional vaccines, and in particular for vaccines against viral infections. For example, two types of influenza vaccines are currently in use. More conventional The vaccine is an inactivated vaccine (containing a dead virus) which is administered by injection, usually into the arm. The second vaccine, called the nasal spray flu vaccine (sometimes called the live attenuated flu vaccine LAIV), was approved in 2003 and contains an attenuated (attenuated) activity that can be administered by a nasal spray. virus. As described by the World Health Organization (WHO), influenza virus types A and B are both common causes of acute respiratory disease. Although both types of viruses cause infectious diseases of considerable morbidity and mortality, influenza B infection is often limited to localized morbidity, but influenza A virus is the main cause of larger infectious diseases (including widespread infectious diseases worldwide). Cause. Influenza viruses are members of the Orthomyxo family of viruses and have a broad host range including humans, horses, dogs, birds and pigs. It is a envelope antisense RNA virus produced in 8 RNA fragments encoding 10 viral proteins. The virus replicates in the nucleus of the infected host cell. Influenza viruses are most dangerous for young individuals and elderly individuals or individuals with reduced immunity. These viruses can proliferate to high titers in eggs, which act as a vehicle for the formation of viruses for the preparation of influenza vaccines.

Influenza A viruses undergo frequent changes in their surface antigens, while influenza B viruses do not change very frequently. The immunity of a virus strain after infection may not completely protect against subsequent antigenic variants. Therefore, a new anti-influenza vaccine must be designed each year to combat a spread of the virus that is likely to trigger the next infectious disease. Therefore, WHO collects annual data based on monitoring of the most prevalent influenza virus strains transmitted among people and recommends influenza vaccine compositions. Currently, vaccines include two subtypes of influenza A virus and one subtype of influenza B virus in vaccines. Vaccines typically protect about 50%-80% of healthy adults against clinical disease.

Subunit vaccines that can be developed by recombinant DNA technology have so far been disappointing because they exhibit only limited immunogenicity. One example is that recent clinical trials of several HIV (human immunodeficiency virus) subunit vaccines have been discontinued, not only because of the limited efficacy of the vaccine, but in some cases, the immune subject demonstrates accelerated disease progression when subsequently exposed to HIV; See, for example, Cohen, Science 264: 1839 (1994); and Cohen, Science 264: 660 (1994). Subunit vaccine and death virus and recombinant live virus vaccine One disadvantage is that although it appears to elicit a strong humoral immune response, it fails to induce protective cell-mediated immunity. The main conclusion of the 1994 International AIDS Conference was that there is still a need for cytotoxic T cell-mediated responses that have so far been lacking in clinical vaccines to prevent or reduce HIV infectivity. In addition, the HIV vaccine tested to date failed to induce immunity at the mucosal surface where the initial HIV infection occurred.

In addition, the only adjuvant approved for use in the United States is the aluminum salt: aluminum hydroxide and aluminum phosphate, none of which stimulates cell-mediated immunity. In addition, aluminum salt formulations cannot be frozen or lyophilized, and such adjuvants are not effective for all antigens.

Yeast cells have been used to produce subunit protein vaccines, including some of those tested in the HIV vaccine trials described above. The yeast can also be fed to the animal for further immune action to trigger an immune response in a non-specific manner (i.e., stimulating phagocytosis and production of complement and interferon). The results are not clear and the protocols do not produce protective cell-mediated immunity; see, for example, Fattal-German et al, Dev. Biol. Stand. 77: 115-120 (1992) and Bizzini et al., FEMS Microbiol. Immunol. 2: 155-167 (1990).

The above studies demonstrate the potential of using recombinant S. cerevisiae as a vaccine and immunotherapeutic vehicle. See, for example, U.S. Patent Nos. 5,830,463 and 7,083,787, and U.S. Patent Publication Nos. 2004-0156858 A1 and 2006-0110755 A1. These yeast-based immunotherapeutic products have been shown to induce immune responses in vivo to kill target cells expressing various viral antigens and cancer antigens in various animal species, and can be antigen-specific, CD8 ⁺ CTL-mediated. Achieve this goal. See also Stubbs et al, Nat. Med. 7: 625-629 (2001) and Lu et al, Cancer Research 64: 5084-5088 (2004). More specifically, other studies have demonstrated that Saccharomyces cerevisiae is greedily phagocytosed by dendritic cells and directly activates dendritic cells, and then delivers yeast-associated proteins to CD4 ⁺ and CD8 ⁺ T in an efficient manner. cell. See, for example, Stubbs et al, Nature Med. 5: 625-629 (2001) and U.S. Patent No. 7,083,787.

In addition to being able to interact directly with dendritic cells, yeast also has a variety of other features that make it an ideal platform for immunotherapy. First, multiple antigens can be designed to be expressed in a single yeast strain (see, for example, Pichuantes et al., "Expression of heterologous gene products in yeast." in Protein Engineering-Principles and Practice, pages 129-162, JLCleland and Edited by CSCraik, Wiley-Liss, New York (1996)). These formulations share a number of advantages with DNA vaccines, including ease of construction and ability to target multiple antigens. Yeast-based immunotherapeutic formulations differ from DNA vaccines in that substantial purification is not required to remove potentially toxic contaminants. The U.S. Food and Drug Administration (FDA) states that yeast is GRAS (known as safe). Thus, the toxicity and safety concerns with other vaccine vectors are not suitable for yeast-based delivery vehicles.

Despite efforts to prepare effective vaccines, there is still a need for vaccine compositions that effectively stimulate various immune responses. For influenza vaccines, the ratio of disease to children, the elderly, and certain high-risk groups is still significant, and in developing countries, vaccination may be occasional or non-existent. In industrialized countries, production problems, the high cost of producing vaccines using current technology, and long-term obstruction of the production of adequate influenza vaccines to supply recipient groups. In addition, the threat of new strains and the possibility of future infectious diseases have increased our interest in the efficient production of influenza vaccines. Therefore, there is a need in the art for improved vaccines that provide long-lasting and effective protection against a variety of influenza strains and that can be rapidly and safely produced for use by humans and other animals. However, these concerns and needs are not only for influenza vaccines, but for other types of vaccines, including vaccines targeting other viruses and other infectious agents.

In fact, many pathogens (including bacteria and parasites) infect individuals in stages, causing the immune system to cope with different antigen subpopulations at various stages. In addition, many pathogens have evolved A series of strategies that allow pathogens to "avoid" or evade the immune system. Finally, like the above viruses, many pathogens elicit antigens and produce antigenic variants, especially those antigens that are expressed or localized on their surface, and can also be infected by pathogens of multiple or multiple viral strains at the same time, all of which The vaccination strategy is complicated. For example, a parasite that causes malaria (for example, Plasmodium falciparum or Plasmodium vivax) is originally infected as a child through the bloodstream due to the bite of the infected mosquito, but The liver cells are then infected very quickly, and the daughter cells undergo a fundamental change in the liver cells to become merozoites. The merozoites are released from hepatocytes and rapidly infect red blood cells, which multiply, differentiate, and continue to infect other cells in red blood cells. Therefore, an ideal vaccine should be able to direct the immune system to recognize and destroy parasites at all stages, whether in the blood, in the liver, or in red blood cells. However, most vaccines do not have the ability to prevent or eliminate all infections, but rather focus on limiting the ability of pathogens to cause disease or toxic to individuals, but other stages of the life cycle and latent infections remain unresolved.

Therefore, for infectious diseases that fight diseases caused by infectious diseases or other agents, it is necessary to have the ability to control or influence the type of immune response induced, such as diseases or conditions that are prevented or treated, and/or individuals are giving The timing point is dependent on the immune status of a particular antigen or pathogen, preferentially inducing a cell-mediated immune response (eg, producing cytotoxic T cells (CTL)), preferentially inducing a humoral response (eg, an antibody response), or inducing both types of immune responses . In addition, compositions are provided which, upon administration of several administrations, elicit an effective immune response and are effective to elicit an immune response upon exposure to low levels of antigen (equivalent dose).

The invention disclosed herein provides compositions and methods that meet the above needs. Immunotherapeutic products (eg, vaccines) based on platform technology for yeast-based vaccines are simple to produce, are not neutralized by host immune responses, can be administered repeatedly to elicit antigen-specific immune responses, and do not require patient-specific manufacturing methods .

One embodiment of the invention relates to a vaccine. In one aspect, the vaccine comprises: (a) a first yeast coal preparation comprising at least one heterologous intracellular antigen; and (b) a second yeast coal preparation comprising at least one heterologous extracellular antigen. In one aspect, the vaccine comprises: (a) a first yeast coal containing at least one heterologous intracellular antigen and at least one heterologous extracellular antigen; and (b) comprising at least one heterologous intracellular antigen or a second yeast coal agent of at least one heterologous extracellular antigen. In another aspect, the vaccine comprises: (a) a yeast coal containing at least one heterologous intracellular antigen and at least one heterologous extracellular antigen; and (b) a non-yeast based composition, It comprises at least one antigen contained in the yeast broth of (a) or an antigen derived from the same pathogen or disease, wherein the non-yeast based composition is selected from the group consisting of a DNA vaccine, a protein subunit vaccine, and death or loss. Live pathogens.

In the above embodiments of the present invention, the yeast coal preparation of (a) may be formulated to be delivered by the same or different administration route as the non-yeast based composition of (b). In one aspect, the intracellular antigen is an antigen that is expressed internally by the pathogen. In one aspect, the extracellular antigen is a structurally conserved antigen of the same type of pathogen. In one aspect, the extracellular antigen is an antigen that appears on the surface of the pathogen. In one aspect, the extracellular antigen is a structurally variable antigen of the same type of pathogen. In one aspect, the antigen is derived from an infectious pathogen.

Another embodiment of the invention relates to a vaccine containing at least one influenza antigen. In one aspect, the vaccine comprises (a) a yeast broth; and (b) an influenza virus fusion protein expressed by a yeast bacterium, the influenza virus fusion protein comprising: an influenza matrix (M1) protein and At least a portion of the influenza ion channel protein (M2) influenza protein. In one aspect, the vaccine comprises (a) a first yeast vehicle that exhibits an influenza virus fusion protein comprising: an influenza matrix (M1) protein, an influenza ion channel protein (M2), and a nucleus At least a portion of an influenza protein of a sheath (NP) protein; and (b) at least one other yeast bacterium that exhibits an influenza virus fusion protein comprising: a hemagglutinin (HA) protein and a ceramide At least a portion of an influenza protein of a phosphatase (NA) protein. In one aspect, the vaccine comprises (a) a yeast vehicle; and (b) is expressed by the yeast agent Influenza virus fusion protein comprising: at least a portion of at least one first influenza protein selected from the group consisting of influenza matrix (M1) protein, influenza ion channel protein (M2), and nuclear sheath (NP) protein; At least a portion of at least one second influenza protein of a hemagglutinin (HA) protein and a neuraminidase (NA) protein. In another aspect, the vaccine comprises (a) a yeast vehicle; and (b) a first influenza virus fusion protein expressed by the yeast agent, the first influenza virus fusion protein comprising a component selected from the group consisting of influenza ( At least a portion of at least one influenza protein of the M1) protein, influenza ion channel protein (M2), and nuclear sheath (NP) protein; and (c) a second influenza virus fusion protein expressed by the yeast vehicle, the second influenza The viral fusion protein comprises at least a portion of at least one influenza protein selected from the group consisting of a hemagglutinin (HA) protein and a neuraminidase (NA) protein. In still another aspect, the vaccine comprises (a) a yeast vehicle; and (b) at least one selected from the group consisting of an influenza matrix (M1) protein, an influenza ion channel protein (M2), and a nuclear sheath (NP) protein. At least a portion of the influenza virus protein; and (c) at least a portion of at least one second influenza protein selected from the group consisting of a hemagglutinin (HA) protein and a neuraminidase (NA) protein.

In the above examples, in one aspect, the HA protein is selected from the group consisting of: H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16. In one aspect, the HA protein is selected from the group consisting of: H1, H2, and H3. In one aspect, the HA protein is H5. In one aspect, the NA protein is selected from the group consisting of: N1, N2, N3, N4, N5, N6, N7, N8, and N9. In one aspect, the NA protein is selected from the group consisting of: N1 and N2. In one aspect, the M1 protein is an intracellular protein relative to the yeast vehicle. In one aspect, the M2 protein is a protein that is intracellular, extracellular, or both relative to the yeast vehicle. In one aspect, the M2 protein is M2e. In one aspect, the NP protein is an intracellular protein relative to the yeast vehicle. In one aspect, the HA protein or NA protein is an extracellular protein relative to the yeast vehicle. In this aspect, the NA protein can also be an intracellular protein relative to the yeast vehicle.

In the above examples, in one aspect, the influenza virus fusion protein comprises an amino acid sequence represented by SEQ ID NO: 1 (MADEAP) at its N-terminus. In one aspect, the flow The susceptible viral fusion protein comprises at least a portion of the Aga2 protein or Cwp2 protein at its N-terminus or C-terminus sufficient to target the fusion protein to the cell wall of the yeast vehicle.

Another embodiment of the present invention includes a vaccine comprising: (a) a yeast vehicle; and (b) an influenza virus fusion protein comprising an M1 antigen, which is expressed as a single cell by yeast vehicle recombination An internal fusion protein consisting of SEQ ID NO:4.

Another embodiment of the present invention includes a vaccine comprising: (a) a yeast vehicle; and (b) an influenza virus fusion protein comprising an H1 antigen, which is expressed as a single cell by yeast vehicle recombination An internal fusion protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 20.

Yet another embodiment of the present invention includes a vaccine comprising: (a) a yeast vehicle; and (b) an influenza virus fusion protein comprising an H1 antigen, which is expressed as a single cell by yeast vehicle recombination An outer fusion protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 36.

Another embodiment of the invention relates to a vaccine comprising: (a) a yeast vehicle; and (b) an influenza virus fusion protein comprising an H5 antigen, the H5 antigen being recombined as a single by yeast vehicle An extracellular fusion protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 24.

A further embodiment of the invention relates to a vaccine comprising: (a) a yeast vehicle; and (b) comprising an M1 antigen, an N1 antigen and four M2e antigens, recombinantly expressed as a single cell by a yeast vehicle An influenza virus fusion protein of an internal fusion protein consisting of SEQ ID NO: 16.

Another embodiment of the present invention relates to a vaccine comprising: (a) a yeast vehicle; and (b) comprising an N1 antigen which is expressed as a single intracellular fusion protein by yeast vehicle and two M2e An influenza virus fusion protein of the antigen consisting of SEQ ID NO:18.

Another embodiment of the present invention relates to a vaccine comprising: (a) a yeast vehicle; and (b) comprising an H3 antigen and a N2 antigen which are recombinantly expressed as a single extracellular fusion protein by a yeast vehicle. Influenza virus fusion protein.

In any of the above embodiments, in one aspect, the expression of the fusion protein is under the control of an inducible promoter. In one aspect of any of the above embodiments, the promoter is selected from the group consisting of CUP1 and TEF2.

In one aspect of any of the above embodiments, the yeast vehicle is selected from the group consisting of a whole yeast, a yeast protoplast spheroid, a yeast cytoplasm, a yeast residue, a yeast cell wall preparation, and a subcellular yeast membrane. Extract or its fraction. In one aspect of the above embodiment, the yeast cell or yeast protoplast spheroid system for preparing the yeast vehicle is transformed with a recombinant nucleic acid molecule encoding the fusion protein such that the fusion protein is composed of yeast cells or yeast Recombinant performance of protoplast spheroids. In one aspect of any of the above embodiments, the yeast vehicle is produced using a yeast cell or a yeast protoplast spheroid recombinantly expressing the fusion protein, the yeast vehicle comprising a yeast cytoplasm, a yeast residue, Yeast cell wall preparation or subcellular yeast membrane extract or a fraction thereof. In one aspect of any of the above embodiments, the yeast vehicle is derived from a non-pathogenic yeast. In one aspect of the above embodiment, the yeast agent is derived from a yeast selected from the group consisting of: Saccharomyces , Schizosaccharomyces , Kluveromyces , Hansenula , Candida , and Pichia . In one aspect of any of the above embodiments, the genus Saccharomyces is Saccharomyces cerevisiae.

In one aspect of any of the above embodiments, the vaccine further comprises dendritic cells in which the yeast vehicle has been intracellularly loaded into the dendritic cells.

In one aspect of any of the above embodiments, the vaccine additionally comprises at least one biological response modifier.

Another embodiment of the invention relates to the preparation of any of the vaccines described herein Use in a formulation that induces an antigen-specific immune response.

Yet another embodiment of the invention relates to the use of any of the vaccines described herein in the preparation of a formulation for protecting an animal against influenza infection.

Another embodiment of the invention relates to the use of any of the vaccines described herein for the preparation of a formulation for inducing an antigen-specific cell-mediated immune response against influenza antigen.

A further embodiment of the invention relates to the use of any of the vaccines described herein in the preparation of a formulation for the treatment or prevention of a disease or condition.

Another embodiment of the invention relates to the use of any of the vaccines described herein in the preparation of a formulation that will immunize a population of individuals at risk of influenza infection.

Another embodiment of the invention relates to the use of any of the vaccines described herein in the preparation of a formulation for treating a population of individuals infected with influenza.

Another embodiment of the present invention relates to a method for producing a yeast-based vaccine (a vaccine comprising a yeast vehicle) described herein, which comprises culturing in a vaccine at a pH greater than pH 5.5. The yeast vehicle.

A further embodiment of the invention relates to a method of producing an influenza vaccine, the method comprising transfecting a yeast vehicle with an influenza antigen fusion protein, wherein the influenza antigen fusion protein comprises at least a portion of an influenza virus protein selected from the group consisting of influenza matrix (M1) protein, influenza ion channel protein (M2), influenza virus nucleocapsid (NP) protein, hemagglutinin (HA) protein, and neuraminidase (NA) protein. In one aspect, the method comprises culturing the yeast vehicle at a pH greater than pH 5.5. Other aspects include: loading the influenza virus protein into the yeast vehicle, mixing the influenza virus antigen with the yeast vehicle, and physically connecting the influenza virus antigen to the yeast vehicle to formulate the transformed yeast The bacterinic vehicle is administered to the individual by injection, or the transformed yeast vehicle is formulated for administration to the individual by intranasal administration.

Another embodiment of the invention relates to a kit for preparing a formulation for inducing a cell-mediated immune response, a humoral immune response, or a combination thereof in an individual, the kit comprising a plurality of yeast vehicles (each of which is a yeast medium) The instructions comprise at least one intracellular heterologous antigen or at least one extracellular heterologous antigen and instructions for preparing the formulation using a yeast vehicle. In one aspect, the yeast vehicle recombinantly expresses the antigen. In one aspect, the kit also includes at least one other composition selected from the group consisting of: (a) a yeast membrane or cell wall particle containing at least one of a heterologous antigen or an antigen from the same pathogen or disease. (b) a yeast vehicle mixed with a heterologous antigen or at least one of the antigens of the same pathogen or disease; (c) DNA encoding at least one of the antigens or antigens from the same pathogen or disease Vaccine; (d) a protein subunit vaccine comprising at least one of the antigens or antigens from the same pathogen or disease, or a protein subunit vaccine comprising an antigen from the same pathogen or disease; and/or (e) inclusion A death pathogen or a passivating pathogen of at least one of the heterologous antigens. In one aspect, the intracellular antigen is an antigen that is expressed internally by the pathogen. In one aspect, the extracellular antigen is a structurally conserved antigen of the same type of pathogen. In one aspect, the extracellular antigen is an antigen that appears on the surface of the pathogen. In one aspect, the extracellular antigen is a structurally variable antigen of the same type of pathogen.

Figure 1 is a schematic diagram illustrating influenza virus and its components. One of the more highly conserved antigens is circled within the circle.

Figure 2 is a digital image of the Western blot method showing the intracellular performance of influenza matrix protein 1 (M1, also known as MP or MP1) in yeast.

Figures 3A and 3B show the results of CTL assays in which mice immunized with a yeast vehicle expressing influenza matrix protein (M1) can intracellularly induce M1-specificity (Fig. 3A) and influenza against target cells that are infected with influenza. Virus specific (Fig. 3B) cytotoxic T cells (CTL).

Figure 4 is a digital image of the Western blot method from the lysate of P815 cells infected with influenza A/PR/8/34, indicating that P815 cells can be infected with influenza virus and can be used in CTL assays. Used as a target cell.

Figure 5 shows the results of a T lymphocyte proliferation assay in which mice immunized with a yeast vehicle expressing influenza matrix protein (M1 or MP) can induce an M1-specific T cell response intracellularly.

Figure 6 shows the results of a T lymphocyte proliferation assay in which mice immunized with a yeast vehicle expressing influenza matrix protein (M1) can induce influenza-specific T cell responses intracellularly.

Figure 7 is a schematic representation of the digital image (top) of the Western blot method and the fusion structure (bottom) illustrating the intracellular performance of influenza hemagglutinin (HA) (Aga2-HA) fused to Aga2 in yeast.

Figure 8 shows the results of a CTL assay in which a mouse vehicle that exhibits influenza hemagglutinin (HA) (Aga2-HA) fused to Aga2 is vaccinated by two different routes of administration to induce intracellular influenza virus. Specific CTL response.

Figure 9 shows the results of T lymphocyte proliferation assay in which mice immunized with two different routes of administration by yeast vehicle expressing influenza hemagglutinin (HA) fused to Aga2 (Aga2-HA) can be intracellularly Induces influenza virus-specific T lymphocyte proliferative response.

Figure 10A depicts the Tarmogen construction protocol, which allows any of the target antigens studied to be expressed on the surface of the yeast vehicle (top panel). The following figure shows the specific Tarmogen, also known as GI-8003, which is a yeast vehicle designed to act as an Aga2-HA fusion protein (shown on its surface) of influenza A/PR/8/34 HA (H1). . The digitalized image of the Western blot method indicated the performance of the Aga2-HA protein.

Figure 10B depicts an exemplary structure in which an antigen is designed to be expressed on the surface of a yeast. The figure schematically illustrates how various yeast proteins can be used as examples of spacer arms.

Figure 11 depicts a Tarmogen (which expresses influenza HA protein on the yeast surface via cell wall protein 2 (cwp2)) expressing a fusion protein (referred to as VK8 in Figure 10B) and is shown with a yeast vehicle alone (GI-1001 or Compared to YVEC), flow cytometry analysis of intact cells The histogram of HA on the surface of the yeast (Tarmogen also known as GI-8000-S).

Figures 12A-12G show histograms in which influenza HA proteins are localized on the surface using various methods (described above and illustrated in Figure 10B). Figure 12A shows the yeast control (YEX) for Figures 12B and 12C. Figures 12B-12C illustrate the performance of the Tarmogen showing VK4 (Figure 12B) and the performance of the Tarmogen showing TK75-15 (Figure 12C). Figure 12D shows the yeast control (YEX) for Figures 12E-12G. Figures 12E and 12F show the performance of HA VK8 by yeast when yeast is glycosylated (Figure 12E) and deglycosylated (Figure 12F). Figure 12G shows that HA is expressed on the plasma membrane of Lu002-expressing protoplast spheroid preparations.

Figures 13A and 13B are schematic diagrams showing the structure of the expression of influenza hemagglutinin (HA) for extracellular (Figure 13B) and intracellular (Figure 13A), which can be correlated with conserved influenza antigens M1, NP and M2 (Figure 13A). Intracellular expression combinations.

Figures 14A and 14B show T cell priming (Figure 14B) and antibody production (Figure 14A) for the three protocols used. Protocol A used only PBS (control). Protocol B was primed on days 0 and 28 with PBS and challenged with soluble ovalbumin (ova) on day 56. Protocol C was primed with a yeast vehicle expressing ovalbumin (OVAX) and challenged with soluble ovalbumin. Figure 14B shows the results of a T cell activation assay using various amounts of soluble ovalbumin to re-excite T cells obtained from mice in vitro, which mice have been rendered immune by each of the three protocols described above. .

Figure 15 shows the results of experiments in which GI-2010 (Tarmogen expressing HIV Gag protein) was tested for its ability to elicit an antibody response (with infection with live vaccinia virus not encoding HIV-Gag protein (control) or encoding HIV-Gag protein (virus) Compared with the observed humoral reaction). The saline curve and the control curve were below the GI-2010 and YVEC curves (i.e., the GI-2010 and YVEC strains were superimposed on the physiological saline curve and the control curve).

Figures 16A and 16B are schematic diagrams showing the combination of signals obtained by the reaction of B cell activation with a helper T cell for peptides from antigens associated with the search for antibodies (signal 1 is shown in Figure 16A and signal 2 is shown in Figure 16B). in).

Figure 17 is a digitalized image showing the cell surface appearance of the influenza HA1 domain (Aga2-HA1) fused to Aga2.

The present invention is generally directed to compositions and methods for effectively inducing various types of immune responses, including cell-mediated immune responses, humoral immune responses, and combinations thereof. The invention is useful for inducing protective and/or therapeutic immune responses against a variety of antigens, including pathogens, and such reactions can be optimized to preferentially induce (or ensure induction) of cell-mediated immune responses, humoral immune responses, or cells. Both mediate and humoral immune responses. Furthermore, the immune response elicited by the vaccine and vaccine strategy of the present invention can be optimized to provide protection against frequent mutations and/or to simultaneously infect individual pathogens with multiple or multiple viral strains, to protect against infection at different life cycle stages, or to exist in Pathogens and defenses in individuals evade the effective response of pathogens of the immune system by a variety of actions, including infection of target cells. The immune response elicited by the present invention is particularly effective not only in situations where the humoral immune response is at least somewhat effective in limiting the ability of the pathogen to cause disease, but in which the immune system does not prevent or detect alternative forms of the pathogen (mutant or life cycle) The form and/or host cell is particularly effective in the case of infection or encroachment by a pathogen (which may then evade or mislead the immune response). Depending on the type of immune response that is most favorable for a particular disease or condition, and depending on the immune status of the patient to a given antigen or pathogen at a given point, the immune response elicited by the vaccine and vaccine strategy of the present invention may also be Optimized to protect or treat individuals with a disease or condition. Finally, the vaccine and vaccine strategy of the present invention can elicit an effective immune response after several administrations, and can also effectively elicit an immune response upon exposure to low levels of antigen (saving dose).

The present invention provides compositions and methods for eliciting immune responses to a variety of antigens suitable for protective or therapeutic immunization and/or vaccination purposes. In one aspect, the invention provides a composition that induces a humoral immune response and a cell-mediated immune response, in other aspects, provides a composition that preferentially induces or triggers a humoral immune response, and a vaccine strategy (including initiation of targeting one) Or the production of antigen-specific antibodies of various antigens studied, or preferentially induced or triggered Compositions and vaccine strategies for cell-mediated (cellular) immune responses, including cytotoxic T cell responses. Priority induction means that the immune response can be directed to or directed to a particular type of immune response, primarily based on how the yeast-based vaccine of the invention can be used to make the antigen available to the immune system. Compositions and immunization strategies can be specifically designed to provide optimization of the immune response as described above. The invention also provides compositions and methods that can enhance or supplement immunogenicity or smooth immunity using non-yeast based vaccines, such as DNA vaccines.

More specifically, the present invention is directed to a yeast-based immunotherapeutic product (as described in U.S. Patent Nos. 5,830,463 and 7,083,787, and U.S. Patent Publication Nos. 2004-0156858 A1 and 2006-0110755 A1). Improvements in platform technology and the provision of novel yeast-based vaccines for inducing protective and/or therapeutic immune responses against a variety of antigens, including pathogens. In one aspect, the invention utilizes a vaccine composition capable of selectively designing a yeast-based vaccine composition to display or localize antigens, or to select different antigens and antigens, either intracellularly, extracellularly (ie, yeast surface representation) or both. Combination for intracellular and/or extracellular expression/localization, for manipulating the type of immune response that preferentially induces defense against a particular antigen, and for manipulating an immune response to most effectively confer immunity to the individual against a particular antigen or antigen and most effectively prevent or The ability to treat a disease or condition associated with an antigen or antigen.

For example, in one embodiment of the invention disclosed herein, the disclosed compositions and methods can provide a cross-protective "universal" vaccine pathway to provide prolonged immunity against an antigen or antigen and, in particular, cell-mediated Guided immunity. This principle of the invention takes advantage of the fact that pathogens, such as certain antigens, are highly conserved among different strains or species of the pathogen. In addition, certain cells targeted by the immune response, such as tumor cells, may also share specific conserved antigens. These antigens are often expressed internally by pathogens or cells (ie, antigens displayed on the surface of the pathogen or cell are more likely to be altered or mutated to evade immunodetection and clearance, and for all strains, all species or all cells) In other words, antigens inside pathogens or cells are more likely to be conserved). Such conservative antigens An effective cell-mediated immune response in which a protoplasm (such as a virus) is expressed internally, which can also be generically referred to as an internal antigen, is cross-protective and capable of inducing a defense antigen (and thereby protecting against a pathogen or a cell encroached by the pathogen). (Cellular) yeast-based vaccines provide the basis. In this aspect, the conserved antigen or internal antigen is typically expressed or provided intracellularly by the yeast vehicle of the invention. However, the performance of such types of antigens is not limited to intracellular expression; conserved antigens or internal antigens may or additionally be expressed or provided extracellularly (on the surface of yeasts) by the yeast vehicle of the invention, as described below.

The intracellular expression or localization of the antigen by the yeast vehicle of the present invention (for example, by intracellular expression, by intracellular loading, or any other method of providing an antigen contained in the intracellular environment of the yeast) It is generally suitable for preferentially inducing a cell-mediated immune response against any antigen, or more specifically, in the context of a susceptible cell-mediated immune response (although humoral immune responses can also be induced or induced by this method, especially If the naturally occurring form of the antigen is present or already present in the extracellular environment (ie, by prior infection, disease or immunity). Intracellular expression or antigen delivery by yeast vehicle is especially useful for antigen-priming or vaccination, as the ability to provide a strong cell-mediated immune response and better immune memory enhances cell-mediated and humoral immune response. In response to future encounters with antigens (eg, by stimulating immunity, infection, or disease). Intracellular expression or provision of an antigen is suitable for inducing an immune response against any antigen, including the above-described conserved antigen or internal antigen and the variable or external antigen described below.

In another embodiment, compositions and methods are provided that are designed to induce a viral strain, species or antigen variant specific immune response. For example, the method can confer immunity to a host against a more specific antigen associated with a particular mutant, strain, strain, or life cycle stage or specific antigenic variant of the pathogen, such as in a conventional death virus vaccine (generally included) This is the case for the three selected strains representing the three viral groups based on surface antigens. In other words, the other facts utilized in this aspect of the invention are that many pathogens and cells exhibit protein (variable antigen) changes, particularly those that can be used to form yeast-based vaccines. A protein change on a pathogen or cell surface (a surface antigen may also generally be referred to herein as an external antigen) that provides a complete direct or even seasonal immunity to the defense antigen or pathogen. In one aspect of the invention, the antigens are typically expressed extracellularly (on the surface) by the yeast vehicle of the invention. The expression of the variable antigen or external antigen is not limited to extracellular representation or extracellular delivery of the antigen to the yeast vehicle; such antigens may or additionally be expressed or provided intracellularly by the yeast vehicle of the invention, as discussed below.

Performing extracellular or superficial expression or providing antigen by the yeast vehicle of the present invention (for example, by attaching an antigen to an outer surface or by secreting an antigen from a yeast, causing surface expression or translocation of the antigen to a yeast vehicle) The expression of the outer surface) preferentially induces a humoral immune response to the defense antigen, or more specifically, enhances the induction of a humoral immune response compared to when the antigen is expressed in the cell by the yeast vehicle, although the cell-mediated immune response is also It can be initiated or induced by this method. In fact, one of the advantages of this vaccine is that the vaccine of the present invention induces cell-mediated defense against such surface antigens as compared to the above-described conventional death virus vaccines (e.g., which primarily induces defense virus neutralizing antibody responses). Immune reaction with body fluids. The extracellular expression by the yeast vehicle or the provision of an antigen is suitable for inducing an immune response against any antigen, including the above-mentioned conserved antigen or internal antigen and variable antigen or external antigen. However, extracellular expression or the provision of an antigen on a yeast vehicle is particularly useful for inducing an immune response against antigens (such as soluble antigens, cell surface antigens or antigens on the surface of pathogens) that the immune system is expected to encounter in the extracellular environment, It is therefore necessary to develop a humoral immune response against these antigens.

In one aspect, the present invention also combines the above two vaccine methods (intracellular and extracellular expression by a yeast vehicle or providing an antigen) to provide a highly efficient novel vaccine effective in inducing cell-mediated immunity and humoral immunity. Novel vaccines can be designed to provide cross-protective immunity and more specific immunity against specific antigenic variants or pathogen species, strains or mutants. For example, with regard to viral infections (such as influenza infections), the combination vaccine approach can cross-protect the "universal" approach along with the virus-specific antigen method to induce a sense of defense against influenza. Effective immune response. This method induces a cell-mediated immune response and a humoral immune response against influenza virus, and in a preferred embodiment of the invention, achieves this goal in a cross-protection manner with a virus strain specific manner.

In this embodiment of the invention, the vaccines can be designed in any of a number of ways. For example, in one aspect, a conserved antigen from a pathogen (eg, a viral internal antigen) can be expressed or provided in a cell by a yeast vehicle, and a variable antigen (eg, a viral surface antigen) from a pathogen can be yeast The bacterial vehicle is expressed or provided extracellularly. Immunization with these yeast agents induces a cell-mediated immune response and a humoral immune response against the virus, and this is achieved in a cross-protective manner with a virus-specific manner. Individuals immunized with a vaccine containing the yeast vehicle have strong cell-mediated immunity against a conserved antigen and strong humoral immunity against a variable antigen, although both types of immune responses are elicited against both types of antigen. How to modify this first instance to improve or modify the immune response to the virus is apparent to those skilled in the art. For example, a conserved antigen and a variable antigen can be expressed or provided by a yeast in a cell, and the variable antigen can be expressed or provided extracellularly by the yeast to ensure a good cell-mediated immune response against both types of antigen ( It is important for eliciting future cell-mediated and humoral immune responses) and provides more effective humoral immunity against variable antigens (see, for example, Figure 16 and discussed herein).

In a combined embodiment described herein, the yeast vehicle that exhibits extracellularly or provides an antigen (on the surface of the yeast vehicle) can be the same or a different yeast than the yeast vehicle that exhibits or provides the antigen within the cell. Fungal media. Furthermore, different combinations of intracellular antigens and/or extracellular antigens can be present on different yeast vehicles, and such vehicles can be used separately or together, depending on the desired vaccination. In general, when the antigen is provided by two or more different yeast agents (i.e., as opposed to providing or providing all antigens in a yeast vehicle), the yeast agents can be combined (mixed) for administration as a single vaccine (for example, a single injection or other type of administration) or a different yeast vehicle can be ordered Dosing. Sequential administration can be intervald at any suitable time, including small time increments (seconds or minutes) and longer time increments (day, week, month, or even year). The present invention contemplates that any combination of antigens (which in a preferred embodiment includes at least one intracellular antigen and at least one extracellular antigen) can be used in such embodiments, and that such antigens can be used to express or provide such antigens. Provided by any combination of yeast vehicles, including single yeast vehicles.

Thus the above vaccine methods can be modified by a combination of different vaccines based on yeast or sequential administration (eg, initiation/initiation strategies), wherein different combinations of antigens (including different combinations of extracellular antigens and/or intracellular antigens) are provided. And, in some aspects, different combinations of conserved antigens and/or variable antigens are provided. In addition, the yeast vehicles described herein can be combined with other types of vaccines simultaneously or sequentially (eg, in initiation and challenge protocols) to further direct the immune response and provide enhanced protection against infection and disease. In one embodiment, a yeast-based vaccine of the invention that provides at least cell-mediated immunity, and a yeast-based vaccine of the invention that provides cell-mediated immunity and humoral immunity in an aspect (such as by a cell) Internal or extracellular expression or antigen supply) is used to elicit an immune response against a specific antigen, antigenic group or pathogen. Then by passing a conventional vaccine (such as a DNA vaccine, a protein subunit vaccine or a dead or inactivated pathogen) or by other vaccines based on yeast (including membrane or cell wall particle vaccine) or yeast and conventional antigen preparations Combining or even yeast alone (e.g., in the latter two cases, the yeast is primarily used as an adjuvant) provides immune challenge. In such "priming-inducing" strategies, the strong cell-mediated response induced by the first immune effect (initiation) improves the efficacy of subsequent stimuli, and in some embodiments may actually provide a synergistic effect, especially when The challenge vaccine is a different type of vaccine than the priming vaccine and/or contains different antigens compared to the priming vaccine.

By eliciting an immune response using the yeast-based vaccines and methods of the invention, cell-mediated and humoral immune memory (i.e., memory B cells and T cells that selectively recognize the antigen of interest) can be produced. Thus, after subsequent exposure to the antigen (eg, via vaccination challenge, disease or infection), the immune system responds more quickly and more efficiently, and Essentially, for vaccination challenge, lower antigen doses can be used in non-yeast based vaccine-based elicitors (see, for example, Example 2 and Figures 14 and 16). In addition, the yeast vehicle can act synergistically with a non-yeast based vaccine to optimize the immune response by a combination approach such as combining a DNA vaccine with a yeast based vaccine. Thus, such vaccines need to be administered only once and/or in lower amounts to achieve efficacy. Such dose-saving properties are required when a non-yeast based vaccine is out of stock or when combating a threat to public health, since it is difficult to impart more than one immunity to an individual, especially in a large number of immune settings.

The compositions and methods of the present invention also include wherein the yeast vehicle does not necessarily recombine or otherwise provide the antigen of interest but is used as an adjuvant to enhance delivery alone or to carry a sufficient amount of antigen to carry a non-antigen upon administration. An individual that induces an immune response following a yeast vehicle, such as an individual who is infected with a pathogen at the time, an individual who undergoes a mutation in a cellular protein, or an antigen that otherwise exhibits or carries an immune system intolerance or an antigen that can break the tolerance of its tolerance. A vaccine for the immune response of an antigen, such as any conventional vaccine, including a DNA vaccine, a subunit protein vaccine, a dead or inactivated pathogen, a dendritic cell vaccine, and the like. As described above, this method can be combined with pre-immunization or subsequent immunization of a yeast vehicle that exhibits or provides an intracellular and/or extracellular heterologous antigen.

In fact, there is a great deal of flexibility in how the vaccine of the invention is designed and used. For example, a "universal" vaccine comprising a yeast vehicle that exhibits or is complexed (ie, associated, mixed, contained, provided) with certain antigens (such as conserved antigens) can be administered to the individual on a regular basis to Cross-protective immunity is produced. This vaccine can then be combined, for example, once or periodically with other yeast agents that exhibit or are complexed with other antigens, such as variable antigens, to treat specific viral strains known to be transmitted in the population of individuals. A yeast vehicle that exhibits or is complexed with such variable antigens may be rotated, alternated, or selected once a year or based on any other preferred option (eg, an emergency or an expected epidemic or pandemic, or otherwise required) Targeting the pathogen of interest and/or the most prevalent during a given period or for a specific geographic area Pathogen virus strain. Other embodiments of the invention will be apparent from the disclosure provided herein.

As described above, the type of expression of the manipulated antigen or the type of association of the antigen with the yeast vehicle can result in specific immune outcomes that can be utilized as needed to "customize" or "design" for a population, individual or specific A vaccine for a disease, condition or pathogenic infection. Humoral immunity and cell-mediated immunity can be induced in a condition in which the yeast vehicle exhibits or provides an antigen outside the cell, although this type of performance provides or induces induction compared to the presence or antigen provided by the yeast in the cell. Humoral immunity is especially effective. The reason for this is mainly that in this embodiment, the antigen is directly exposed to B cells, allowing B cell activation, proliferation, maturation, and antibody production to occur more efficiently.

More specifically, an antigen expressed or provided on the surface of a yeast (or secreted by a yeast) can be recognized by a B cell antigen receptor (BCR) expressed by B cells (B lymphocytes). After the surface antigen binds to BCR, the B cell then internalizes the yeast agent that expresses the antigen-binding antigen, and the antigen is treated to be antigen-derived from a major histocompatibility complex (MHC) class II receptor. The peptide form is returned to the B cell surface. The MHC-peptide complexes are bound by "helper" T cells (eg, CD4 ⁺ T cells) that have a T cell receptor (TCR) that specifically recognizes a particular MHC-peptide complex. Activation of antigen-specific T cells that recognize MHC-peptide complexes presented by B cells can in turn lead to B cell proliferation and its progeny differentiation and maturation into signalling forms of antibody-secreting cells (eg, cytokines) to such B cells provide help". Helper T cells can be contacted by T cells with the B cells that are presenting the MHC-antigen complex and by MHC-peptide complexes presented by other antigen presenting cells, including dendritic cells and macrophages. activation. Furthermore, since the yeast vehicle of the present invention is greedily phagocytosed by the MHC class I channel (except the MHC class II channel) of antigen presenting cells such as dendritic cells and directly activates the channel, in addition to the CD4 ⁺ cell mediated reaction CD8 ⁺ cell-mediated immune responses can also be induced by extracellular manifestations or by providing antigens. In this way, humoral immune responses and cell-mediated immune responses can be induced by the presence or absence of antigens by the yeast, and it is believed that this type of performance is more likely to induce a humoral immune response than by intracellular expression or antigen supply. effective. Humoral immune responses can include the production of neutralizing antibodies suitable for the prevention and treatment of infectious diseases and other undesirable conditions.

A schematic representation of B cell activation that produces a humoral immune response is shown in Figures 16A and 16B. Referring to Figure 16A, for signal 1 to activate B cells, the antigen may be provided by a non-yeast based vaccine or soluble protein, or by a yeast that exhibits, presents or otherwise contains a target antigen on the surface. Referring to Figure 16B, intracellular treatment is presented via an MHC class 2 receptor by the antigenic system contained in the activated B cell, and the antigen-specific helper T cell recognizes and is activated by the antigen. Activation of signals and cytokines transmitted by antigen-specific T cells allows B cells to mature and stimulate antibody production. The yeast expressing the target antigen in the cell is extremely effective in activating the antigen-specific helper T cells and amplifying the number of antigen-specific helper T cells, as described above and below. Antibody production can be induced more efficiently when helper T cell activation and proliferation occurs before or in conjunction with B cell binding of soluble target antigen.

When the antigen is expressed or provided by the yeast vehicle in the cell, the cell mediates the immune response (CD4 ⁺ reacts with CD8 ⁺ T cells) by the antigen via MHC of antigen presenting cells (such as dendritic cells and macrophages) Class I restricted channels are presented in conjunction with MHC class II restricted channels, as described above (see, for example, U.S. Patent Nos. 5,830,463 and 7,083,787, Stubbs et al., Nat. Med. 7:625-629 (2001) and Lu. Et al, Cancer Research 64: 5084-5088 (2004)). T cells activated by this mechanism also contribute to the production of antibodies, for example, by providing signals to B cells that encounter target antigens (such as non-yeast based vaccines) via different routes or by, for example, natural exposure to pathogens or diseases. Experimental results demonstrating this concept are shown in Figures 14 and 15.

Various aspects of the invention may be better understood by the specific examples of influenza vaccines disclosed herein, although the invention is not limited to such vaccines. Indeed, in conjunction with the information provided herein, those skilled in the art utilize manipulation, conserved antigens, and variable antigens (or internal antigens) that exhibit or provide antigen in the cell and/or in the cell by the yeast vehicle of the present invention. The manipulation of the expression or provision of the external antigen) and the manipulation of the triggering and stimulating methods described herein can readily extrapolate the vaccine strategy described for influenza viruses to other pathogens and immunization protocols (where the target antigen is a cellular protein).

In one embodiment, the invention is generally directed to novel compositions and methods for vaccinating an animal to prevent influenza virus and to treat or prevent influenza infection in an animal. The invention includes the use of a combination of a yeast-based vaccine or a yeast-based vaccine comprising at least one yeast vehicle and at least one influenza antigen selected to induce an immune response against influenza infection in the animal. In a particularly preferred embodiment, the invention encompasses the use of a combination of influenza antigens in a yeast-based vaccine wherein the combination of antigens provides protection against cross-protection of various influenza virus strains as well as protection against specific strains of particular virus strains. The invention provides a novel yeast-based vaccine for preventing influenza virus infection by using a cross-protective "universal" vaccine method alone or in conjunction with a strain-specific antigen method. This method can induce a cell-mediated immune response and a humoral immune response against influenza virus, preferably in a cross-protection manner and a virus-specific manner.

In the first aspect of this embodiment, the present invention utilizes the fact that certain proteins expressed internally by influenza viruses are highly conserved among viral strains. Such antigens (generally referred to herein as internal viral proteins) are the basis for yeast-based vaccines that are cross-protective and that elicit an effective cell-mediated immune response against viral proteins (and thus influenza virus-infected cells). In other aspects of this embodiment, the present invention utilizes the fact that influenza virus strains exhibit changes in surface proteins (generally referred to herein as external viral proteins) that can be used to form more specific immune defenses for the host. A yeast-based vaccine of a viral strain, as in a conventional lethal virus vaccine that typically includes three selected strains representing three surface antigen-based viral groups. However, the vaccines of the present invention induce cell-mediated and humoral immune responses against such viral surface antigens as compared to such conventional death virus vaccines, which primarily induce defense virus neutralizing antibody responses. Furthermore, the present invention combines the two vaccines in a preferred aspect to provide cross-protection Novel and effective influenza vaccines that are immune to viral strain-specific immunity, including cell-mediated immunity and humoral immunity.

common technology

The practice of the present invention may utilize, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology well known to those skilled in the art. Such techniques are described in detail, for example, in Methods of Enzymology , Vol. 194, Guthrie et al., Cold Spring Harbor Laboratory Press (1990); Biology and activities of yeasts , by Skinner et al., Academic Press (1980); Methods in yeast genetics: a laboratory course manual , Rose et al, Cold Spring Harbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle and Cell Biology , Pringle et al., Cold Spring Harbor Laboratory Press (1997); The Yeast Saccharomyces : Gene Expression , Jones et al., Cold Spring Harbor Laboratory Press (1993); The Yeast Saccharomyces: Genome Dynamics, P rotein Synthesis, and Energetics , Broach et al., Cold Spring Harbor Laboratory Press (1992); Molecular Cloning: A Laboratory Manual , Second Edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual , Third Edition (Sambrook and Russel, 2001), (abbreviated herein as "Sambrook"); Current Protocols in Molecular Biology (FMAusubel, etc.) Edited by human, 1987, including the entire 2001 supplement); PCR: The Polymerase Chain Reaction , (Mullis et al, ed., 1994); Harlow and Lane (1988) Antibodies, A Laboratory Manual , Cold Spring Harbor Publications, New York; Harlow and Lane (1999) Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (herein referred to herein as "Harlow and Lane"), edited by Beaucage et al., Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000); Casarett and Doull's Toxicology The Basic Science of Poisons , edited by C. Klaassen, Sixth Edition (2001), and Vaccines , S. Plotkin and W. Orenstein, eds., Third Edition (1999).

General definition

"Humoral immune response" generally refers to all processes of antibody production and concomitant antibody production, including but not limited to B lymphocyte (B cell) activation, affinity maturation, differentiation into plasma cells and memory B cell production, germinal center formation and isomorphism And T helper cell activation, signaling and cytokine production, as well as antibody effector functions, including neutralization, typical complement activation and opsonization.

"Cell-mediated" immune response (which is used interchangeably herein with the term "cell" immune response) generally refers to including T lymphocytes (including cytotoxic T lymphocytes (CTL)), dendritic cells, giant The response of immune cells of phagocytes and natural killer cells to antigens, and all processes accompanying such reactions, including but not limited to activation and proliferation of such cells, CTL effector functions, effects involving adaptive immune responses and innate immune responses The cytokine production of other cells functions as well as the production of memory T cells.

According to the invention, the term "extracellular" (as used herein to provide an antigen outside the cell relative to a yeast vehicle) means that the antigen obtainable by any of a variety of methods occurs in a yeast vehicle. Extracellular (superficial or external). For example, if the antigen is expressed by a yeast vehicle (eg, by recombinant production) such that the antigen or a portion thereof is present (located, contained, localized) on the surface of the yeast vehicle (eg, intact yeast) On the cell wall or the protoplast spheroids of the yeast, the plasma membrane of the cytoplasm and the residue, the antigen is extracellular with respect to the yeast vehicle. The antigen may, for example, be expressed in the ER of the yeast and then translocated to the surface of the yeast, although in general, relative to the production of recombinant antigen, reference is made to "express" on the surface of the yeast vehicle or in the yeast vehicle. Extracellular "expression" is intended to encompass the entire process of antigen production, from transcription, via translation, via antigen targeting and delivery or translocation to the final destination in yeast vehicle. The term "performance" is also generally used interchangeably with the term "providing" and most generally covers any means of providing an antigen on the surface of a yeast vehicle. (ie, covering a combination of other methods). For example, if an antigen is attached to the outer surface of the yeast, such as by covalent or non-covalent bonds (ie, not necessarily by yeast recombination), the antigen is also extracellular relative to the yeast vehicle. . If the antigen is only mixed with the yeast (mixture, combined, composition), the antigen is also extracellular relative to the yeast vehicle. If the antigen is secreted by the yeast, the antigen is also extracellular relative to the yeast vehicle.

According to the invention, the term "intracellular" (as used herein to provide an antigen in a cell relative to a yeast vehicle) means that the antigen obtainable by a plurality of methods is contained within the intracellular environment of the yeast vehicle. . For example, if the antigen is expressed by a yeast vehicle (eg, by recombinant production) and at least a portion of the antigen produced remains in the yeast (ie, not translocated or delivered to the yeast vehicle) The surface is either not delivered or translocated to the surface of the yeast vehicle, and the antigen is intracellular relative to the yeast vehicle. The intracellular environment of a yeast vehicle can include, but is not limited to, cytosol, ER, endome, and secretory vesicles that have not yet traversed to the cell surface. If the antigen (eg, by any suitable delivery method, including electroporation, particle bombardment, microinjection, lipofection, adsorption, infection, and protoplast fusion) is loaded into the yeast, the antigen is also relative to the yeast vehicle. For intracellular.

According to the invention, the term "antigen" as used herein generally refers to: any part of a protein (peptide, partial protein, full-length protein) in which the protein is naturally occurring or derived from synthesis; cell composition (intact cells, cells) Lysates or dividing cells); organisms (intact organisms, lysates or dividing cells) or carbohydrates (such as those expressed on cancer cells), or other molecules, or a portion thereof. The antigen induces an antigen-specific immune response (eg, a humoral and/or cell-mediated immune response) of the same or similar antigen encountered within the cells and tissues of the individual to which the antigen is administered. Alternatively, the antigen can act as a tolerogen.

When referring to a stimulatory immune response, the term "antigen" is used interchangeably with the term "immunogen." An immunogen as used herein, describes a condition that induces a humoral and/or cell-mediated immune response (ie, is antigenic) such that the immunogen (eg, via the vaccine of the invention) Administration to an animal can elicit an antigen that protects against the antigen-specific immune response of the same or similar antigen encountered within the animal tissue.

"Tolerance" is used to describe an antigen that causes a reduced or altered immune response to the antigen and preferably responds to contact with the tolerogen or to present or present the cells of the tolerance. Substantially non-responsive, non-reactive, other forms of inactivation or deletion of immune system cells, amounts or routes of administration are provided.

The "vaccination antigen" can be not only an immunogen or a tolerogen, but also an antigen used in a vaccine in which a biological reaction (inducing an immune response, tolerance limit) is induced against a vaccinated antigen.

An "immunogen domain" of a given antigen can be any portion, fragment or epitope (eg, a peptide fragment or subunit or antibody epitope) that contains at least one antigen that acts as an antigenic determinant when administered to an animal. Other conformational epitopes). For example, a single protein can contain multiple different immunogenic domains. The immunogenic domain is not necessarily a linear sequence within the protein, such as in the context of a humoral immune response.

An epitope is defined herein as a single immunological in situ point within a given antigen sufficient to elicit an immune response, or a single tolerogenic in situ point within a given antigen sufficient to inhibit, eliminate, or inactivate an immune response. . Those skilled in the art will appreciate that T cell epitopes differ in size and composition from B cell epitopes, and epitopes presented via class I MHC channels differ from antigens presented via class II MHC channels. base. The epitope can be a linear sequence or a conformational epitope (conserved binding region). The antigen may be generally small, or larger, as a single epitope, and may include multiple epitopes. Thus, the antigen size can be as small as about 5-12 amino acids (eg peptides) and can be as large as full length proteins, including multimers and fusion proteins, chimeric proteins, intact cells, intact microorganisms or parts thereof (eg intact cells) The dissolved product or the extract of the microorganism). In addition, the antigen can include a carbohydrate that can be loaded into a yeast vehicle or loaded into a composition of the invention. It will be appreciated that in some embodiments (ie, when the antigen is derived from a yeast medium from a recombinant nucleic acid molecule) When present, the antigen is a protein, a fusion protein, a chimeric protein or a fragment thereof, rather than an intact cell or microorganism. The influenza fusion proteins of the invention described herein are preferred.

"vaccination" or "immunization" refers to an immune response that induces (induces) a defense antigen or an immunogenic portion or a tolerogenic portion thereof by administering an antigen alone or in combination with an adjuvant. Vaccination preferably produces a protective or therapeutic effect, wherein subsequent exposure to the antigen (or source of the antigen) induces an immune response against the antigen (or source) that slows or prevents the disease or condition of the animal. The concept of vaccination is well known in the art. The immune response elicited by administration of the composition (vaccine) of the present invention may be in any aspect of the immune response (eg, cell-mediated response, humoral response, cytokine production) as compared to non-administration of the composition. Any detectable change.

Tarmogen (target molecule) refers broadly to a yeast vehicle that expresses one or more heterologous antigens either extracellularly (on its surface), intracellularly (internal or intracellular) or extracellularly and intracellularly. Tarmogens have generally been described in this technology. See, for example, U.S. Patent No. 5,830,463.

In one embodiment of the invention, any of the amino acid sequences described herein may be combined with at least one and up to about 20 other heterologous amino acids, the other heterologous amino acids being located in a particular amino acid sequence. The sides of each of the C-terminus and/or the N-terminus. The resulting protein or polypeptide may be referred to as "primarily comprising" a particular amino acid sequence. As described above, according to the present invention, the heterologous amino acid is an amino acid sequence which is flanked by a specific amino acid sequence, which is not naturally occurring (that is, does not exist in nature, in vivo); or with a specific amino acid sequence a functionally unrelated amino acid sequence; or an amino acid sequence not encoded by a nucleotide flanking the naturally occurring nucleic acid sequence encoding a particular amino acid sequence (when it is present in the gene) (if a standard code is used) The sub-selection method translates the nucleotides of the naturally occurring sequence of the organism from which the amino acid sequence is derived. Similarly, the phrase "consisting essentially of", when used in connection with a nucleic acid sequence as referred to herein, refers to a nucleic acid sequence encoding a particular amino acid sequence, the nucleic acid sequence The flanks are at least 1 and up to about 60 other heterologous nucleotides, and the other heterologous nucleotides are located at the 5' and/or 3' ends of the nucleic acid sequence encoding the particular amino acid sequence. A heterologous nucleotide located on the side of a nucleic acid sequence encoding a particular amino acid sequence (when it is present in a native gene) is not naturally occurring (ie, not found in nature, in vivo), or does not encode a protein for any other A protein that functions or alters the function of a protein having a particular amino acid sequence.

According to the invention, a "heterologous amino acid" is an amino acid sequence which is flanked by a particular amino acid sequence, non-naturally occurring (i.e., not found in nature, in vivo); or functions with a particular amino acid sequence An unrelated amino acid sequence; or an amino acid sequence not encoded by a nucleotide located on the side of a naturally occurring nucleic acid sequence encoding a particular amino acid sequence (when it is present in the gene) (if standard codon usage is used) The method translates the nucleotides of the naturally occurring sequence of the organism from which the amino acid sequence is derived. Thus, at least two amino acid residues heterologous to the influenza antigen are any two non-naturally occurring amino acid residues flanking the influenza antigen.

According to the present invention, the phrase "selectively binds" refers to the ability of an antibody, an antigen-binding fragment of the invention, or a binding partner to preferentially bind to a particular protein. More specifically, the phrase "selectively binds" refers to the specific binding of a protein to other proteins (eg, antibodies, fragments thereof or antigen binding partners), by any standard assay (eg, immunoassay) The measured binding amount was statistically significantly higher than the background control used for the assay. For example, when performing an immunoassay, the control group typically includes a reaction well/tube containing only an antibody or antigen-binding fragment (ie, lacking an antigen), wherein the amount of reactivity of the antibody or antigen-binding fragment lacking the antigen ( For example, a non-specific binding reaction well) is considered a background. Binding can be measured using various standard methods in the art, including enzyme immunoassays (eg, ELISA), immunoblot assays, and the like.

"Individual" is a vertebrate, a better mammal, and a better human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice, and rats. Term The body "can be used interchangeably with the term "animal", "subject" or "patient".

As used herein, "selected from the group consisting of" means any one of the group of listed items or any combination of the plurality of listed items. Thus, for example, "items selected from the group consisting of A, B, and C" may also be expressed as "items selected from A, B, and/or C."

An isolated protein or polypeptide as referred to in the present invention includes a full length protein, a fusion protein, or any fragment, domain, conformation epitope or homolog of such protein. More specifically, according to the present invention, an isolated protein is a protein (including a polypeptide or peptide) that has been removed from its natural environment (ie, has been manipulated by humans) and may include, for example, purified protein, partially purified protein, recombinant production Proteins and synthetically produced proteins. Thus, "isolation" does not reflect the extent to which the protein has been purified. The isolated protein of the present invention is preferably produced recombinantly. In accordance with the present invention, the terms "modification" and "mutation" are used interchangeably, and in particular, the modification/mutation of the amino acid sequence (or nucleic acid sequence) of a protein or a portion thereof described herein.

As used herein, the term "homolog" is used to mean that it differs from a naturally occurring protein or peptide (ie, a "prototype" or "wild type" protein) by slightly modifying a naturally occurring protein or peptide, However, proteins or peptides that maintain a naturally occurring form of a basic protein and a side chain structure. Such modifications include, but are not limited to, modifications of one or more amino acid side chains; modifications of one or more amino acids, including deletions (eg, truncation of proteins or peptides), insertions and/or substitutions; Or stereochemical modification of several atoms; and/or several derivatizations including, but not limited to, methylation, glycosylation, phosphorylation, acetylation, myristylation, prenylation, palmitate, Addition of amidoxime and/or glycosylphospholipids to inositol. A homolog has enhanced, reduced or substantially similar properties compared to a naturally occurring protein or peptide. Homologs may include agonists of proteins or antagonists of proteins. Homologs can be prepared using techniques known in the art for the preparation of proteins, including but not limited to: direct modification of isolated naturally occurring proteins, direct synthesis of proteins, or modification of encoded proteins using, for example, typical or recombinant DNA techniques. Nucleic acid sequences to achieve random mutations or targeted mutations.

A homologue of a given protein may comprise or consist essentially of an amino acid sequence with a reference protein The columns are at least about 45% identical or at least about 50% identical or at least about 55% identical or at least about 60% identical or at least about 65% identical or at least about 70% consistent or at least about 75% consistent or at least about 80% consistent or at least About 85% identical or at least about 90% identical or at least about 95% uniform or at least about 96% consistent or at least about 97% uniform or at least about 98% consistent or at least about 99% consistent (or in full increments of 45) Amino acid sequence of any percentage consistency between % and 99%). In one embodiment, the homolog comprises or consists essentially less than 100% identical to the naturally occurring amino acid sequence of the reference protein, less than about 99% identity, less than about 98% identity, less than about 97% identity, Amino acid sequences having less than about 96% identity, less than about 95% identity, and the like (in increments of 1% to less than about 70% identity).

As used herein, reference to percent (%) identity, as used herein, refers to the assessment of homology using the following: (1) BLAST 2.0 Basic BLAST homology search with standard default parameters (used Blastp searches for amino acids and uses blastn to search for nucleic acids), where a query sequence for filtering low complexity regions is scheduled (described in Altschul, SF, Madden, TL, Schääffer, AA, Zhang, J., Zhang, Z., Miller, W . & Lipman, DJ (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25: 3389-3402, which is incorporated herein by reference in its entirety); BLAST 2 alignment (using the following parameters); (3) and/or PSI-BLAST (Position-Specific Iterated BLAST) with standard default parameters. It should be understood that due to the difference in standard parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific sequences with significant homology can be identified using the BLAST 2 program, and one of the sequences is used as the query sequence, BLAST 2.0 Basic BLAST Performing a search may not identify the second sequence that best matches. In addition, PSI-BLAST provides an automated, easy-to-use version of the "profile" search, which is a sensitive way to find sequence homologs. The program first performs a gap BLAST database search. The PSI-BLAST program constructs a specific site scoring matrix using any significant alignment information returned, and replaces the query sequence for the next round of database searches. Therefore, you should understand the percentage consistency you can use Any program in the program is determined.

Two specific sequences can be aligned with each other using BLAST 2 sequences, as described in Tatusova and Madden, (1999), "Blast 2 sequences-a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250. The document is hereby incorporated by reference in its entirety. A BLAST 2.0 algorithm was used to perform a gap BLAST search between two sequences (BLAST 2.0), and a BLAST 2 sequence alignment was performed with blastp or blastn, and gaps (deletions and insertions) can be introduced in the resulting alignment. For the purposes of this disclosure, BLAST 2 sequence alignments were performed using the standard default parameters as follows.

For blastn, use 0 BLOSUM62 matrix: match reward=1

Mismatch penalty = 2

Open vacancies (5) and extended vacancies (2) penalties

Vacancy x attenuation (50) expected (10) word size (11) filtering (enabled)

For blastp, use 0 BLOSUM62 matrix: open vacancy (11) and extended vacancy (1) penalty

Vacancy x attenuation (50) expected (10) word size (3) filtering (enabled).

An isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural environment (i.e., has been manipulated by humans), the natural environment of which is the genome or chromosome in which the naturally occurring nucleic acid molecule is located. Thus, "isolation" does not necessarily reflect the extent to which a nucleic acid molecule has been purified, but indicates that the molecule does not include the complete genome or intact chromosome in which the naturally occurring nucleic acid molecule is located. An isolated nucleic acid molecule can include a gene. An isolated nucleic acid molecule comprising a gene is not a fragment of a chromosome comprising the gene, but a fragment comprising a coding region and a regulatory region associated with the gene, but no other genes naturally occurring on the same chromosome. An isolated nucleic acid molecule can also include a specific nucleic acid sequence (ie, a heterologous sequence) that is flanking (ie, at the 5' end and/or 3' end of the sequence) that is otherwise not normally flanked by a particular nucleic acid sequence. . Isolating nucleic acid molecules can include DNA, RNA (eg, mRNA), or DNA or RNA derivatives. Biological (eg, cDNA). Although the phrase "nucleic acid molecule" refers primarily to a solid nucleic acid molecule and the phrase "nucleic acid sequence" refers primarily to the sequence of nucleotides on a nucleic acid molecule, the two phrases are used interchangeably, especially for encoding proteins or proteins. A nucleic acid molecule or nucleic acid sequence of a domain.

A recombinant nucleic acid molecule is any nucleic acid sequence operably linked to at least one sequence of any transcription control sequence capable of effectively modulating the expression of a nucleic acid molecule in a cell to be transfected, encoding any one or more of the proteins described herein. At least one sequence of molecules. Although the phrase "nucleic acid molecule" refers primarily to a solid nucleic acid molecule and the phrase "nucleic acid sequence" refers primarily to the sequence of nucleotides on a nucleic acid molecule, the two phrases are used interchangeably, particularly for nucleic acids capable of encoding a protein. Molecular or nucleic acid sequence. Furthermore, the phrase "recombinant molecule" refers primarily to a nucleic acid molecule operably linked to a transcriptional control sequence, but can be used interchangeably with the phrase "nucleic acid molecule" administered to an animal.

A recombinant nucleic acid molecule comprises a recombinant vector, which is any nucleic acid sequence, typically a heterologous sequence, operably linked to an isolated nucleic acid molecule encoding a fusion protein of the invention, which enables recombinant production of the fusion protein and which is capable of The invention delivers a nucleic acid molecule into a host cell. The vector may contain a non-naturally occurring nucleic acid sequence adjacent to the isolated nucleic acid molecule to be inserted into the vector. The vector may be RNA or DNA, prokaryotic or eukaryotic, and is preferably a virus or a plastid in the present invention. Recombinant vectors can be used to select, sequence, and/or manipulate nucleic acid molecules and can be used to deliver such molecules (eg, in DNA vaccines or viral vector-based vaccines). Recombinant vectors are preferably used to represent nucleic acid molecules and may also be referred to as expression vectors. The recombinant vector is preferably capable of being expressed in a transfected host cell.

In the recombinant molecule of the present invention, the expression vector operation of the nucleic acid molecule with a regulatory sequence (such as a transcription control sequence, a translation control sequence, an origin of replication, and other regulatory sequences compatible with the host cell and controlling the expression of the nucleic acid molecule of the present invention) Sexual connection. In particular, recombinant molecules of the invention include nucleic acid molecules operably linked to one or more expression control sequences. The phrase "operably linked" refers to the transfection of a nucleic acid molecule (ie, transformation, conversion) The manner in which the nucleic acid molecule is linked to the expression control sequence when expressed or transfected into the host cell.

According to the invention, the term "transfection" is used to mean any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell. The term "transformation" is used interchangeably with the term "transfection" (when the term is used to refer to the introduction of a nucleic acid molecule into a microbial cell, such as algae, bacteria, and yeast). In the microbial system, the term "transformation" is used to describe a genetic modification of an exogenous nucleic acid obtained by a microorganism, and is basically synonymous with the term "transfection." Thus, transfection techniques include, but are not limited to, transformation, chemical processing of cells, particle bombardment, electroporation, microinjection, lipofection, adsorption, infection, and protoplast fusion.

Vaccines and compositions of the invention

Embodiments of the invention relate to a composition (vaccine) useful in a method for eliciting a cell-mediated and/or humoral immune response against an antigen or antigens, in a preferred embodiment protecting the animal Prevent disease or condition (including pathogen infection) or alleviate at least one symptom caused by the disease or condition. Such compositions generally comprise: (a) a yeast vehicle; and (b) a heterologous antigen expressed by the yeast vehicle in association or combination with the yeast vehicle. Other compositions may include a yeast vehicle in combination with a heterologous antigen provided in the form of other vaccine compositions, such as DNA vaccines, protein subunit vaccines, or dead or inactivated pathogens. When the yeast vehicle exhibits one or more antigens, the antigen can be expressed or provided in any combination, intracellularly, extracellularly, or both. In certain embodiments, the antigens are provided as a fusion protein designed to stabilize the performance of the heterologous protein in the yeast vehicle, to prevent subsequent translation of the heterologous protein expressed, and/or in In some embodiments, the fusion protein can be expressed on (including translocated to) the surface of the yeast vehicle (extracellular expression). Fusion proteins also provide a broad range of cell-mediated immune responses and, in some embodiments, humoral immune responses, preferably more than one different antigen, and/or can be combined with other yeast agents that exhibit different antigens. Although one or more of such fusion proteins can be loaded into a yeast vehicle (eg, as an egg as described herein) White matter) or otherwise complexed or mixed with a yeast vehicle to form a vaccine of the invention is an embodiment of the invention, but such fusion proteins are most typically composed of yeast vehicles (eg, whole yeast or yeast protoplasts) The body, which may be further processed as a yeast cytoplast, yeast residue or yeast membrane or cell wall extract or fraction or particles thereof, as a recombinant protein.

According to the present invention, a heterologous fusion protein, a "heterologous" protein expressed by a yeast means that the protein is not a protein naturally expressed by the yeast, although the fusion protein may comprise a yeast sequence or a protein naturally expressed by the yeast or Part of it (for example, the Aga protein described herein). For example, a fusion protein of influenza hemagglutinin protein and yeast Aga protein is considered to be a heterologous protein suitable for use in the present invention because the fusion protein is not naturally expressed by yeast.

Yeast vehicle

In any of the compositions (e.g., vaccines) of the present invention, the following aspects relating to yeast vehicles are encompassed by the present invention. According to the invention, a yeast vehicle is any yeast cell (eg whole or intact cell) or derivative thereof (see below) or for use in combination with one or more antigens of a vaccine or therapeutic composition of the invention (see below) or Adjuvant. Thus yeast vehicles can include, but are not limited to, live whole yeast microorganisms (ie, yeast cells having all of its constituents (including cell walls)), dead intact yeast microorganisms, or derivatives thereof, including: yeasts Protoplast spheroids (ie, yeast cells lacking cell walls), yeast cytoplasmic bodies (ie, yeast cells lacking cell walls and nucleus), yeast residues (ie, yeast lacking cell walls, nucleus, and cytoplasm) Bacterial cells), subcellular yeast membrane extracts or fractions thereof (also known as yeast membrane particles and previously referred to as subcellular yeast particles) or yeast cell wall preparations.

Yeast protoplast spheroids are typically produced by enzymatic digestion of yeast cell walls. This method is described, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194, 662-674., which is incorporated herein in its entirety by reference.

Yeast cytoplasts are usually produced by enucleating yeast cells. This method is described, for example, in Coon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55, which is herein incorporated by reference in its entirety.

Yeast residues are typically produced by resealing permeabilized or solubilized cells and may, but need not, contain at least a portion of the organelles of the cells. Such methods are described, for example, in Franzusoff et al, 1983, J. Biol. Chem. 258, 3608-3614 and Bussey et al, 1979, Biochim. Biophys. Acta 553, 185-196, each of which is incorporated herein by reference in its entirety.

Yeast membrane particles (subcellular yeast membrane extracts or fractions thereof) refer to yeast membranes that lack natural nuclear or cytoplasm. The particles can be of any size, including sizes ranging from natural yeast membranes to ultrasonic treatment or other membrane splitting methods known to those skilled in the art, followed by re-sealing of the particles. A method for preparing a subcellular yeast membrane extract is described, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194, 662-674. It is also possible to use an antigen to be isolated from the yeast membrane particles containing the yeast membrane portion and (when the antigen is recombined by the yeast, and the yeast membrane particles are prepared). The antigen can be carried inside the membrane, on the membrane surface, or a combination thereof (ie, the antigen can be located inside and outside the membrane and/or across the membrane of the yeast membrane particles). In one embodiment, the yeast membrane particles are recombinant yeast membrane particles, and the recombinant yeast membrane particles may be intact, split or split and resealed yeast membranes comprising at least a membrane surface An antigen or antigen is at least partially embedded in the membrane.

An example of a yeast cell wall preparation is an antigen carried on its surface, or an antigen at least partially embedded in the interior of the cell wall such that the yeast cell wall preparation stimulates the desired (eg, protective) immune response to prevent infectious agents when administered to the animal. Yeast cells are isolated.

Any yeast strain can be used to prepare the yeast vehicle of the present invention. The yeast is a single-cell microorganism belonging to one of three categories: Ascomycetes, Basidiomycetes, and Fungi Imperfecti. Selected as an immunomodulator A major consideration for the type of yeast is the pathogenicity of the yeast. In one embodiment, the yeast is a non-pathogenic virus strain, such as Saccharomyces cerevisiae. Selection of a non-pathogenic yeast strain minimizes any adverse effects on the individual to which the yeast vehicle is administered. However, if the pathogenicity of the yeast can be eliminated by any means known to those skilled in the art, pathogenic yeasts (e.g., mutant strains) can be used. Although a pathogenic yeast strain or a non-pathogenic mutant thereof has been used in the past as an adjuvant or as a biological response modifier, and can be used according to the present invention, it is preferably a non-pathogenic yeast strain.

Preferred genera of yeast strains include Saccharomyces (Saccharomyces), Candida species (Candida) (which can be pathogenic), the genus Cryptococcus (Cryptococcus), the genus Hansenula yeast (Hansenula), CFR Kluyveromyces , Pichia , Rhodotorula , Schizosaccharomyces , and Yarrowia , more preferably yeast Genus, Candida, Hansenula, Pichia and Schizosaccharomyces, and particularly preferably Yeast. Preferred species of yeast strains include Saccharomyces cerevisiae , Saccharomyces carlsbergensis , Candida albicans , Candida kefyr , Candida tropicalis ( Candida tropicalis ), Cryptococcus laurentii , Cryptococcus neoformans , Hansenula anomala , Hansenula polymorpha , crispy Kluvi Yeast ( Kluyveromyces fragilis ), Kluyveromyces lactis , Kluyveromyces marxianus var.lactis , Pichia pastoris , deep red yeast Rhodotorula rubra , Schizosaccharomyces pombe , and Yarrowia lipolytica . It should be understood that a wide variety of such sub-species, types, subtypes, etc., are intended to be included within the above species. More preferred yeast species include Saccharomyces cerevisiae, Candida albicans, Hansenula polymorpha, Pichia pastoris, and Schizosaccharomyces pombe. Saccharomyces cerevisiae is particularly preferred because it is relatively easy to handle and is "recognized as safe" or "GRAS" for use as a food additive (GRAS, FDA Rule 65FR18938, filed April 17, 1997). An embodiment of the invention is a yeast strain capable of replicating a plastid to a particularly high number of replicates, such as a Saccharomyces cerevisiae strain. The Saccharomyces cerevisiae strain is a strain capable of supporting a performance vector that allows one or more target antigens and/or antigen fusion proteins to be expressed in high amounts. Furthermore, any mutant yeast strain can be used in the present invention, including those exhibiting reduced expression modifications after the target antigens are expressed, such as mutations in enzymes that continue N-linked glycosylation.

In one embodiment, the preferred yeast vehicle of the present invention is capable of fusing with a cell type that is delivering a yeast vehicle and antigen, such as dendritic cells or macrophages, thereby effecting a yeast vehicle (in the case of The antigens in many embodiments are efficiently delivered to cell types. As used herein, fusion of a yeast vehicle to a target cell type means that the yeast cell membrane or its particles are capable of fusing with a membrane of a target cell type (eg, dendritic cells or macrophages) to form a syncytium. As used herein, syncytia are polynuclear groups of protoplasts produced by pooling cells. Many viral surface proteins (including those of HIV, influenza, polio, and adenovirus-deficient viruses) and other fusions (such as those involved in the fusion between eggs and sperm) have been shown to work. Fusion between two membranes (ie, between a virus and a mammalian cell membrane or between mammalian cell membranes). For example, a yeast vehicle that produces an HIV gp120/gp41 heterologous antigen on its surface is capable of fusing with CD4+ T-lymphocytes. However, it should be understood that the incorporation of the targeting moiety into the yeast vehicle is not required in some circumstances, but is not required. This may be a further advantage of the yeast vehicle of the present invention in the case where the yeast vehicle exhibits an antigen outside the cell. It has been demonstrated above that the yeast vehicle of the present invention is readily accepted by dendritic cells (as well as other cells such as macrophages).

Yeast vector preparation methods and methods for expressing yeast vehicles, combining or associating yeast vehicles with antigens are described below.

antigen

The antigen to be used in the present invention includes any antigen which is desired to induce an immune response. For example, such antigens may include, but are not limited to, any antigen associated with a pathogen, including viral antigens, fungal antigens, bacterial antigens, helminth antigens, parasitic antigens, ectoparasite antigens, protozoal antigens, or any other infection. Antigen of the substance. The antigen may also include any antigen associated with a particular pathogen or condition (whether from a pathogenic source or a source of cells) including, but not limited to, cancer antigens, autoimmune disease related antigens (eg, diabetes antigens), allergic antigens ( An allergen), a mammalian cell molecule harboring one or more mutated amino acids, a protein typically expressed by the mammalian cell during its prenatal or nascent life, which exhibits a protein induced by the insertion of an epidemiological vector (eg, a virus), It expresses proteins induced by gene translocations and proteins whose expression is induced by mutations in regulatory sequences. The antigens can be native antigens or genetically engineered antigens modified in some manner (eg, sequence modification or production of fusion proteins). It will be appreciated that in some embodiments (i.e., when the antigen is expressed by a yeast vehicle derived from a recombinant nucleic acid molecule), the antigen can be any epitope, fusion protein or chimeric of the protein or its immunogenic domain. Protein, not intact cells or microorganisms.

Other preferred antigens included in the compositions (vaccines) of the invention include those which are capable of inhibiting unwanted or deleterious immune responses (such as due to allergens, autoimmune antigens, inflammatory mediators, antigens involved in GVHD, certain Antigens of cancer, septic shock antigens, and immune responses elicited by antigens involved in transplant rejection. Such compounds include, but are not limited to, antihistamines, cyclosporins, corticosteroids, FK506, peptides corresponding to T cell receptors involved in the production of deleterious immune responses, Fas ligands (ie, binding to cellular Fas receptors) An appropriate MHC complex, a T cell receptor, and an autoimmune antigen present in an extracellular or intracellular domain, a compound that induces apoptosis, in a manner that achieves tolerance or non-reactivity, preferably and can enhance or A combination of biological response modifiers that inhibit cell-mediated immunity and/or humoral immunity.

Tumor antigens (cancer antigens) suitable for use in the present invention may include tumor antigens such as proteins from tumor cells, glycoproteins or surface carbohydrates, epitopes derived from tumor antigens, intact tumor cells, mixtures of tumor cells, and parts thereof (eg dissolved product).

In one aspect, the antigen is derived from a virus, including but not limited to: adenovirus, grit virus, bunyaviruses, coronavirus, coxsackie viruses, cytomegalovirus, Ai-ba Epstein-Barr viruses, flaviviruses, hepadnaviruses, hepatitis viruses, herpes viruses, influenza viruses, lentiviruses, measles viruses, mumps viruses, mucoviruses, oncogenic viruses, Orthomyxoviruses, papillomaviruses, Lactobacillus virus, parainfluenza virus, paramyxovirus, parvovirus, picornavirus, poxvirus, rabies virus, respiratory syncytial virus, reovirus, baculovirus, rubella virus, chlamy virus and chickenpox virus. Other viruses include T-lymphocyte viruses such as human T-lymphocyte viruses (HTLV, such as HTLV-I and HTLV-II), bovine leukemia virus (BLVS), and feline leukemia virus (FLV). Lentiviruses include, but are not limited to, human (HIV, including HIV-1 or HIV-2), monkey (SIV), feline (FIV), and canine (CIV) immunodeficiency viruses.

In other aspects, the antigen is derived from an infectious agent derived from a genus selected from the group consisting of Aspergillus , Bordatella , Brugia , Candida species (Candida), chlamydia (Chlamydia), coccidia genus (coccidia), Cryptococcus (Cryptococcus), the genus heartworm (Dirofilaria), the genus Escherichia (Escherichia), Escherichia Francis (to Francisella ), the genus Neisseria gonorrhoeae (Gonococcus), Histoplasma (Histoplasma), Leishmania genus (of Leishmania), Mycobacterium (Mycobacterium), mycoplasma (Mycoplasma), Paramecium genus (of Paramecium), pertussis genus ( pertussis), Plasmodium (Plasmodium), pneumococcus (pneumococcus), Pneumocystis genus (Pneumocystis), genus Rickettsia (Rickettsia), salmonella (Salmonella), Shigella (Shigella), Staphylococcus aureus genera (Staphylococcus), Streptococcus (Streptococcus), Toxoplasma (Toxoplasma), cholera genus (Vibriocholerae), Yersinia (Yersinia). In one aspect, the infectious agent is selected from the group consisting of Plasmodium falciparum or Plasmodium vivax .

In one aspect, the antigen is derived from a bacterium from the family of the following fungi: Enterobacteriaceae, Micrococcaceae, Vibrionaceae, Pasteurella Pasteurellaceae, Mycoplasmataceae, and Rickettsiaceae. In one aspect, the bacterium belongs to the genus selected from the subordinate categories: Pseudomonas (of Pseudomonas), the genus Bordetella (Bordetella), Mycobacterium (Mycobacterium), Vibrio (Vibrio), Bacillus (Bacillus), Salmonella (Salmonella), Escherichia Francis (Francisella), Staphylococcus (Staphylococcus), Streptococcus (Streptococcus), the genus Escherichia (Escherichia), Enterococcus genus (Enterococcus), Bass Deshi genus (Pasteurella) and Yersinia (Yersinia). In one aspect, the bacterium is from a species selected from the group consisting of Pseudomonas aeruginosa , Pseudomonas mallei , and Pseudomonas pseudomallei . , Bordetella pertussis , Mycobacterium tuberculosis , Mycobacterium leprae , Francisella tularensis , Vibrio cholerae , anthrax spores Bacillus anthracis , Salmonella enteric , Yersinia pestis , Escherichia coli , and Bordetella bronchiseptica .

According to the invention, an antigen suitable for use in the composition or vaccine may comprise two or more immunogenic domains or epitopes from the same antigen, two or more antigens from the same cell, tissue or organism. , immunogenic domain or epitope, or two or more antigens, immunogenic domains from different cells, tissues or organisms Or an epitope.

As mentioned above, the fusion proteins used in the vaccines and compositions of the invention comprise at least one influenza antigen for use in vaccinating animals. The composition or vaccine may include one, two, several, several or a plurality of influenza antigens as desired, including one or more immunogenic domains of one or more influenza antigens.

When an antigen from a pathogen, such as an influenza virus, is used, those skilled in the art can make a yeast vehicle comprising a pathogen antigen by selecting an antigen from a region of the pathogen genome that is highly conserved for different strains of the pathogen. Maximize the long-term efficacy of the vaccine. In addition, the ability of a vaccine to treat a particular infectious disease can be maximized, for example, by selecting an antigen from a region of a variable pathogen, such as an antigen that is different from the virus strain or an antigen that is mutable in each season or geographic region. This aspect of the invention has been discussed in detail above.

In one aspect, the pathogen is an influenza virus. In this aspect, the conserved antigen or internal expression antigen includes: matrix protein (M1), ion channel (M2) antigen, nuclear sheath (NP) antigen, polymerase PB1 (PB1) antigen, polymerase (PB2) antigen, and polymerization. Enzyme PA (PA) antigen. A variable antigen or externally expressed antigen includes a hemagglutinin (HA) antigen (any one or more subtypes) and a neuraminidase (NA) antigen (any one or more subtypes), and an extracellular portion of M2 ( Called M2e). Such antigens can be selected for use in the novel vaccine strategies of the present invention as described above.

In one aspect, the pathogen is a hepatitis virus, such as hepatitis C virus (HCV). In this aspect, the conserved antigen or internal expression antigen includes HCV core protein, HCV NS2, NS3, NS4, NS5. Variable antigens or externally expressed antigens include HCV E1 and E2 (envelope proteins). Such antigens can be selected for use in the novel vaccine strategies of the present invention as described above.

In one aspect, the pathogen is a hepatitis virus, such as hepatitis B virus (HBV). In this aspect, the conserved antigen or the internal expression antigen includes: a core antigen HbcAg and an e antigen HbeAg. Variable antigens or externally expressed antigens include HbsAg (42 nM virions and 22 nM particles). Such antigens can be selected for use in the novel vaccine strategies of the present invention as described above.

In one aspect, the pathogen is an immunodeficiency virus, such as the human immunodeficiency virus (HIV). In this aspect, the conserved antigen or internal expression antigen includes: Vif, Vpr, Nef, p7, nuclear sheath. Variable antigens or externally expressed antigens include gp120 and gp41. Such antigens can be selected for use in the novel vaccine strategies of the present invention as described above.

In some embodiments, the antigen is a fusion protein. In one aspect of the invention, the fusion protein can comprise two or more antigens. In one aspect, the fusion protein can include two or more immunogenic domains of one or more antigens or two or more antigenic determinants (eg, influenza Ml sequence and influenza HA sequence). The vaccine provides antigen-specific immunity in a wide range of patients. For example, a multi-domain fusion protein suitable for use in the present invention may have a plurality of domains, wherein each domain comprises a peptide derived from a specific protein comprising at least 4 amino acid residues on both sides and including the presence A mutant amino acid in a protein, wherein the mutation is associated with a particular disease or condition (eg, a flu infection of a particular strain).

In one embodiment, the fusion protein used as a component of the yeast-based vaccine of the present invention is produced using a structure that is particularly useful for expressing a heterologous antigen in a yeast. Typically, the desired antigenic protein or peptide is fused at its amino terminus to: (a) a specific synthetic peptide that stabilizes the expression of the fusion protein in a yeast vehicle or prevents translation of the fusion protein after expression (the peptides) In particular, for example, U.S. Patent Publication No. 2004-0156858 A1 (published on Aug. 12, 2004), which is hereby incorporated by reference in its entirety herein; Providing a significantly enhanced stability of the performance of the protein in the yeast and/or preventing the protein from being modified by subsequent translation of the yeast cells (these proteins are also described in detail in, for example, U.S. Patent Publication No. 2004-0156858 A1 (supra)) And/or (c) rendering the fusion protein at least a portion of the yeast protein on the surface of the yeast (eg, the Aga protein described in more detail herein).

Furthermore, a fusion peptide or protein can provide an epitope that is designed to be recognized by a selection agent, such as an antibody, and appears to have no adverse effect on the immune response against the vaccinated antigen in the structure. ring. These agents are useful for identifying, selecting and purifying proteins suitable for use in the present invention.

Furthermore, the invention encompasses the use of fusions with the C-terminus of an antigen-encoding structure, particularly for the selection and identification of peptides of proteins. Such peptides include, but are not limited to, any synthetic peptide or natural peptide, such as a peptide tag (eg, 6X His) or any other epitope short tag. The peptide linked to the C-terminus of the antigen of the present invention may be used with or without the addition of the above N-terminal peptide.

A fusion construct suitable for use in the present invention is a fusion protein comprising: (a) at least one antigen (including a full length antigen and immunogenic domains and epitopes of various fusion proteins and multiple antigenic structures as described elsewhere herein) And; (b) synthetic peptides.

In one embodiment, the synthetic peptide is linked to the N-terminus of the influenza antigen, the peptide comprising at least two amino acid residues heterologous to the influenza antigen, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or Prevent translation of the expressed fusion protein after translation. The synthetic peptide and the N-terminal portion of the antigen together form a fusion protein having the following requirements: (1) the amino acid residue at position 1 of the fusion protein is methionine (ie, the first amine in the synthetic peptide) The base acid is methionine; (2) the amino acid residue at position 2 of the fusion protein is not glycine or lysine (ie, the second amino acid in the synthetic peptide is not sweet) (amino acid or proline); (3) the amino acid residues at positions 2-6 of the fusion protein are not methionine (ie, if the synthetic peptide is shorter than 6 amino acids, a portion of a synthetic peptide or a portion of a protein, the amino acid at positions 2-6 does not include methionine; and (4) the amino acid at positions 2-6 of the fusion protein is not lysine Or arginine (ie, if the synthetic peptide is shorter than 5 amino acids, the amino acid at positions 2-6 does not include lysine or arginine, whether part of the synthetic peptide or part of the protein) . The synthetic peptide may be as short as two amino acids, but more preferably at least 2-6 amino acids (including 3, 4, 5 amino acids), and may be longer than 6 amino acids, up to about 200 amino acids, 300 amino acids, 400 amino acids, 500 amino acids or 500 amino acids or more.

In one embodiment, the fusion protein comprises the amino acid sequence MX ₂ -X ₃ -X ₄ -X ₅ -X ₆ , wherein M is methionine; wherein X ₂ is in addition to glycine, lysine, and Any amino acid other than aminic acid or arginine; wherein X ₃ is any amino acid other than methionine, lysine or arginine; wherein X ₄ is methionine-free, Any amino acid other than aminic acid or arginine; wherein X ₅ is any amino acid other than methionine, lysine or arginine; and wherein X ₆ is methionine-free, Any amino acid other than aminic acid or arginine. In one embodiment, the X ₆ residue is a proline. Exemplary synthetic sequences that enhance the stability of influenza antigen expression in yeast cells and/or prevent translation of proteins after translation in yeast include the sequence MADEAP (SEQ ID NO: 1). MADEAP sequences can be used with influenza antigens as well as other antigens. In addition to the increased stability of the performance product, the fusion partner appears to have no adverse effect on the immune response against the vaccinated antigen in the structure. In addition, synthetic fusion peptides can be designed to provide an epitope that can be recognized by a selection agent, such as an antibody.

In other embodiments of the invention, the nucleic acid encoding the translation initiation site for the synthetic peptide of the invention is ACCATGG according to the Kozak translation sequence rule, wherein the ATG in the sequence is the initial translation initiation site and encodes MADEAP (metheine of SEQ ID NO: 1). It will be appreciated that embodiments of the invention as described herein may also be combined. For example, in one aspect of the invention, when the synthetic peptide is MA-D-E-A-P (SEQ ID NO: 1), the nucleic acid encoding the initiation site of the peptide may be A-C-C-A-T-G-G. Various other combinations of embodiments of the invention will be apparent to those skilled in the art.

In one aspect of the invention, the yeast vehicle is manipulated such that the antigen is partially or completely expressed or provided on the surface of the yeast vehicle by delivery or translocation of the expressed antigen product (extracellular which performed). One method of accomplishing this aspect of the invention uses spacer arms to position one or more antigens on the surface of the yeast vehicle. A method of using a spacer arm is to form a fusion protein of the antigen to be studied and a protein that targets the antigen to the cell wall of the yeast. For example, one protein that can be used is a yeast protein (eg, cell wall protein 2 (cwp2), Aga2, Pir4, or Flo1 protein) that enables the antigen to target the yeast cell wall such that the antigen localizes to the surface of the yeast. Other proteins than yeast proteins can be used for the spacer arm; however, for any spacer protein, it is most desirable to have an immunogenic response to the target antigen rather than the spacer arm protein. Thus, if the spacer arm uses other proteins, then The spacer arm protein should not produce this strong immune response to the spacer arm protein itself, so that the immune response against the target antigen is inferior. Those skilled in the art should work on a weak immune response to the spacer protein (relative to the immune response against the target antigen). Any method known to determine the size of an immune response can be used (e.g., antibody production, dissolution assays, etc.) and is readily known to those skilled in the art.

Other methods of locating a target antigen to be exposed on the surface of the yeast use a signal sequence, such as a glycosylphosphatidylinositol (GPI), to anchor the target antigen to the yeast cell wall. Alternatively, localization can be achieved by ligation of the antigen under investigation into the signal sequence of the secretory pathway via translocation into the endoplasmic reticulum (ER) such that the antigen binds to a protein (eg, cwp) that binds to the cell wall.

In one aspect, the spacer protein is a yeast protein. The yeast protein may comprise between about two and about 800 amino acids of the yeast protein. In one embodiment, the yeast protein is between about 10 and 700 amino acids. In other embodiments, the yeast protein is from about 40 to 600 amino acids. Other embodiments of the invention comprise a yeast protein of at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, at least 400 amino acids, at least 450 amino acids, at least 500 Amino acid, at least 550 amino acids, at least 600 amino acids or at least 650 amino acids. In one embodiment, the yeast protein is at least 450 amino acids in length.

In other embodiments, the yeast protein stabilizes the expression of the fusion protein in a yeast vehicle, prevents post-translational modification of the expressed fusion protein, and/or targets the fusion protein to a particular metabolic region within the yeast (eg, Expressed on the surface of yeast cells). Exemplary yeast proteins that can be used for delivery into a yeast secretion channel include, but are not limited to, Aga (including but not limited to Aga1 and/or Aga2); SUC2 (yeast convertase); alpha factor signal leader sequence; CPY; Cwp2p localized and retained in the cell wall; confined to the BUD gene at the yeast cell bud during the initial phase of daughter cell formation; Flo1p; Pir2p; and Pir4p.

In other aspects of the invention, other sequences may be used to target, retain, and/or stabilize the protein to other portions of the yeast vehicle, such as in cytosol or mitochondria. Examples of suitable yeast proteins that can be used in any of the above examples include, but are not limited to, SEC7; phosphoenolpyruvate carboxykinase PCK1, phosphoglycerate kinase PGK, and phosphoric acid for inhibiting expression and intracellular localization in glucose The glyco-isomerase TPI gene product; heat shock proteins SSA1, SSA3, SSA4, SSC1, whose expression is induced and whose protein is more heat-stable after the cells are exposed to heat treatment; mitochondrial protein CYC1; ACT1 for introduction into mitochondria.

For eliciting an effective humoral immune response, the target antigen should be partially expressed or provided on the surface of the yeast (or secreted by the yeast). As shown in Figures 10A and 10B, Figure 11 and Figure 13B and the examples, there may be multiple variations in the expression or provision of antigen on the surface of yeast cells. Those skilled in the art will appreciate that other combinations of yeast proteins can be used to localize one or more of the antigens of interest to the surface.

Those skilled in the art can optimize the performance of the antigen or provide it on the surface of the yeast vehicle in a number of ways. One way to monitor and/or control antigen surface performance. One possible way to achieve this is to optimize the amount of antigen expression to achieve maximum impact. For some antigens, the overexpression of the antigen is toxic to the yeast or to the immune cells and immune system of the individual. In other conditions, too little surface performance results in suboptimal induction of the immune system due to the lack of antigens that interact with B cells. Those skilled in the art can monitor the performance of the antigen by using well known techniques such as flow cytometry (e.g., FACS) and correlating the amount of expression to cell viability.

Other methods of optimizing antigen surface performance or providing are careful selection of spacer arms from cell wall fusion partners. Although examples of yeast proteins that can be used as spacer arms are given below and also shown in Figure 10B, the size of the spacer arms can affect the extent of antigen exposure to the surface of the yeast. Thus, depending on the antigen being used, those skilled in the art should select spacer arms that are suitably spaced apart from the antigen on the surface of the yeast. In an embodiment, the interval The arm is a yeast protein of at least 450 amino acids.

Other considerations for optimizing antigen surface performance are whether the antigen and spacer arm combination should act as a monomer (eg, HA-cwp2 as shown in Figure 11) or a dimer or trimer (eg, a trimeric HA-aga2p attachment) Secreted soluble HA) or even more common linker units. The use of monomers, dimers, trimers, etc., may permit proper spacing or folding of the antigen such that some, if not all, of the antigen is present on the surface of the yeast vehicle in a manner that is more immunogenic. Upper (if for example, the polymeric form employs the configuration required to induce a particular class of antibodies (eg, neutralizing antibodies)).

Those skilled in the art can grow yeast cells (with and without the expression of heterologous antigens) on their surface by incubating the yeast cells at a pH level above 5.5 (i.e., neutral pH). The efficiency in the cytosol is optimized. Neutral pH helps optimize antigen availability and surface presentation, allowing the yeast cell wall to be more compliant and triggering immune cell binding to yeast to produce an optimal immune response including secretion of beneficial cytokines (eg INF-γ) And optimize the activation reaction.

Other methods that can be used by the skilled artisan to optimize the yeast vehicle used to elicit the antibody response are to control the amount of yeast glycosylation. The amount of yeast glycosylation affects the immunogenicity and antigenicity of the antigens present on the surface, as the sugar portion tends to be bulky. Thus, when practicing the present invention, the sugar moiety present on the surface of the yeast and its effect on the three-dimensional space surrounding the target antigen should be considered. Any method can be used to reduce the amount of yeast glycosylation. For example, yeast mutants (eg, mnn1, och1, and mnn9 mutants) that are selected to have low glycosylation can be used or can be cleared by mutating the glycosylation receptor sequence on the target antigen. Alternatively, a yeast having a simplified glycosylation type such as Pichia can be used. An example of the effect of glycosylation on surface antigens is provided in Example 5.

Other considerations for providing antigens on the surface of yeast are how to inactivate the yeast and its potential effect on how to affect the antigenicity of the antigens presented on the surface. The heat inactivation of yeast is the standard way to inactivate yeast, but heat inactivation may change the target. Secondary, tertiary or quaternary structure of the antigen. If heat inactivation is used, those skilled in the art should take care to monitor structural changes in the target antigen by standard methods known in the art. Alternatively, other methods of inactivating yeast, such as chemical methods, electrical methods, radioactive methods, or UV methods, may be used. See, for example, the methods disclosed in standard yeast culture textbooks (such as Methods of Enzymology, Vol. 194, Cold Spring Harbor Publishing (1990)). Any optimization strategy used should take into account the secondary, tertiary or quaternary structure of the target antigen and maintain the structure in order to optimize its immunogenicity.

Other specific aspects of the fusion protein constructs of the invention that are similar to the above examples and which may include the limitations of the above examples (although this is not required) include vaccines comprising a peptide linked to the C-terminus of the influenza antigen, the peptide comprising At least two amino acid residues heterologous to the influenza antigen, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents post-translational modification of the expressed fusion protein. In an exemplary aspect of the invention, the peptide comprises the amino acid sequence E-D (Glu-Asp). This sequence acts to counteract hydrophobicity.

In one embodiment, a vaccine of the invention may comprise a peptide linked to the C-terminus of an influenza antigen, wherein the peptide permits recognition of the fusion protein by an antibody against the peptide. In one aspect, the peptide comprises the amino acid sequence G-G-G-H-H-H-H-H-H (SEQ ID NO: 2). This embodiment can be used alone or in combination with other aspects of the fusion protein described above.

In one embodiment, the yeast protein/peptide, spacer or synthetic peptide for use in the fusion proteins herein comprises an antibody epitope for use in identifying and purifying the fusion protein. Antibodies that selectively bind endogenous antigens are available or can be readily produced. Finally, if it is desired to direct the protein to a particular cellular location (eg, into the secretory pathway, into the mitochondria, into the nucleus), the construct can use endogenous signals for the yeast protein to ensure optimal cellular mechanisms for the delivery system. These signals have been described in more detail above. Preferably, the anti-system of the selective binding partner is purchased or made.

Preparation of yeast vehicle, Tarmogens and composition (vaccine) of the invention

According to the invention, the term "yeast vehicle-antigen complex" or "yeast-antigen complex" "General" is used to describe any association between a yeast vehicle and an antigen. The association includes yeast (recombinant yeast) expression antigen, antigen introduction into yeast, antigenic entity attachment to yeast, and yeast and antigen. Mix together in a buffer or other solution or formulation. These types of complexes are detailed below.

In one embodiment, the yeast cells used to prepare the yeast vehicle are transfected with a heterologous nucleic acid molecule encoding an antigen such that the antigen is expressed by the yeast cells. The yeast is also referred to herein as a recombinant yeast or recombinant yeast vehicle. The yeast cells can then be loaded into the dendritic cells as intact cells, or the yeast cells can be killed, or can be derived, for example, by the formation of yeast protoplast spheroids, cytoplasts, residues or subcellular particles. The derivative is then loaded into the dendritic cells. Yeast protoplast spheroids can also be directly transfected with recombinant nucleic acid molecules (e.g., protoplast spheroids made from whole yeast, and then transfected) to produce recombinant protoplast spheroids that exhibit antigen.

In one aspect, the yeast cell or yeast protoplast spheroid system used to prepare the yeast vehicle is transfected with a recombinant nucleic acid molecule encoding the antigen such that the antigen is recombined by the yeast cell or yeast protoplast spheroid . In this aspect, a yeast vehicle is prepared using a yeast cell or a yeast protoplast spheroid recombinantly expressing an antigen, the yeast agent comprising a yeast cytoplasm, a yeast residue or a yeast membrane particle or a yeast Cell wall particles or fractions thereof.

In general, yeast vehicles and antigens can be associated by any of the techniques described herein. In one aspect, the yeast vehicle is loaded with antigen in the cells. In another aspect, the antigen is attached to the yeast vehicle in a covalent or non-covalent manner. In yet another aspect, the yeast vehicle and the antigen are associated by mixing. In another aspect, and in a preferred embodiment, the antigen is recombinantly expressed by a yeast vehicle or a yeast cell or yeast protoplast spheroid from which the yeast vehicle is derived.

The preferred number of antigens produced by the yeast vehicle of the present invention is the number of any antigen suitably produced by the yeast vehicle, and usually ranges from at least 1 to at least About 6 or more, more preferably from about 2 to about 6 heterologous antigens.

The performance of the antigen in the yeast vehicle of the present invention can be accomplished using techniques known to those skilled in the art. Briefly, a nucleic acid molecule encoding at least one desired antigen is inserted into a performance vector in such a manner that the nucleic acid molecule is operably linked to a transcriptional control sequence such that its characterization or regulation can be achieved when the nucleic acid molecule is transferred into a host yeast cell. Sexual performance. A nucleic acid molecule encoding one or more antigens can be located on one or more expression vectors operably linked to one or more expression control sequences. Particularly important expression control sequences are those that control the initiation of transcription, such as promoters and upstream activation sequences. Any suitable yeast promoter can be used in the present invention, and such various promoters are known to those skilled in the art. Preferred promoters for expression in Saccharomyces cerevisiae include, but are not limited to, promoters encoding the following yeast protein genes: alcohol dehydrogenase I (ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK) ), triose phosphate isomerase (TPI), translation elongation factor EF-1 α (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as TDH3 for triose phosphate dehydrogenase), Galactose kinase (GAL1), galactose-1-phosphate uridine transferase (GAL7), UDP-galactose epimerase (GAL10), cytochrome c ₁ (CYC1), Sec7 protein (SEC7) and acidity Phosphatase (PHO5), more preferably a hybrid promoter (such as the ADH2/GAPDH and CYC1/GAL10 promoters), even more preferably an ADH2/GAPDH promoter (when the glucose concentration in the cell is low (eg, about 0.1 to About 0.2%) was induced) as well as the CUP1 promoter and the TEF2 promoter. Similarly, many upstream activation sequences (UAS) (also known as enhancers) are known. Preferred upstream activation sequences that are expressed in S. cerevisiae include, but are not limited to, UAS encoding genes for the following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1, GAL7, and GAL10, among others. The UAS activated by the GAL4 gene product is particularly preferably ADH2 UAS. Since the ADH2 UAS is activated by the ADR1 gene product, it is preferred to overexpress the ADR1 gene when the heterologous gene is operably linked to the ADH2 UAS. Preferred transcription termination sequences expressed in S. cerevisiae include the termination sequences of the alpha-factor, GAPDH and CYC1 genes.

Preferred transcriptional control sequences for the expression of genes in Methylotrophic yeast include transcriptional control regions encoding genes for alcohol oxidase and formate dehydrogenase.

Optimization considerations and methods for expressing antigens outside the cell by yeast have been detailed above.

Transfection of a nucleic acid molecule into a yeast cell of the invention can be accomplished by any method of introducing a nucleic acid molecule into a cell, including but not limited to diffusion, active delivery, water bath ultrasonic treatment, electroporation, microinjection, lipofection , adsorption and protoplast fusion. The transfected nucleic acid molecule can be incorporated into the yeast chromosome or maintained on an extrachromosomal vector using techniques known to those skilled in the art. Examples of yeast vehicles carrying such nucleic acid molecules are disclosed in detail herein. As described above, yeast cytoplasts, yeast residues, and yeast membrane particles or cell wall preparations can also be produced by transfecting intact yeast microorganisms or yeast protoplast spheroids with desired nucleic acid molecules, and then producing antigens and then using The microorganisms or spheroplasts are further manipulated by techniques known to those skilled in the art to produce recombinantly produced cytoplast, residue or subcellular yeast membrane extracts or fractions thereof containing the desired antigen.

Effective conditions for preparing a recombinant yeast vehicle and expressing the antigen by the yeast vehicle include an effective medium in which the yeast strain can be cultured. Effective media are typically aqueous media containing sources of absorbable carbohydrates, nitrogen and phosphate, as well as suitable salts, minerals, metals and other nutrients such as vitamins and growth factors. The medium may comprise a complex nutrient or may be a defined minimal medium. The yeast strains of the invention can be cultured in a variety of containers including, but not limited to, bioreactors, conical flasks, test tubes, microtiter dishes, and Petri dishes. The culture is carried out at a temperature, pH and oxygen content suitable for the yeast strain. Such culture conditions are well known to those of ordinary skill in the art (see, for example, Guthrie et al. (eds.), 1991, Methods in Enzymology, Vol. 194, Academic Press, San Diego).

In some aspects of the invention, and particularly when it is desired that the antigen has sufficient surface performance in embodiments in which it is desired to induce a humoral immune response, the yeast can be maintained The medium is cultured in a pH level of at least 5.5, that is, the pH of the medium is not allowed to decrease to below pH 5.5. In other aspects, the yeast is maintained at a pH level maintained at about 5.5. In other aspects, the yeast is maintained at a pH level maintained at about 5.6, 5.7, 5.8, or 5.9. In other aspects, the yeast is maintained at a pH level maintained at about 6. In other aspects, the yeast is maintained at a pH level maintained at about 6.5. In other aspects, the yeast is maintained at a pH level maintained at about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In other aspects, the yeast is maintained at a pH level maintained at about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. pH levels are critical for cultivating yeast. Those skilled in the art should be aware that the culture process includes not only the cultivation of yeast but also the maintenance of culture. Since it is known that yeast culture changes to acidity over time (i.e., pH drops), it is necessary to pay attention to monitoring the pH level during the cultivation process. Yeast cell culture in which the pH level of the medium is reduced to below 6 is still encompassed within the scope of the invention, provided that the pH of the medium can be at least 5.5 at some point during the cultivation process. Thus, the longer the yeast is cultured in a medium having a pH of at least 5.5 or above 5.5, the result is better in obtaining a yeast having the desired characteristics.

The use of a neutral pH in the culture of yeast enhances several biological effects that are desirable for the use of yeast as a vehicle for immunomodulation. In one aspect, culturing the yeast at neutral pH allows the yeast to grow well without any adverse effects on cell generation time (eg, slowing down the doubling time). Yeasts can grow to high density without losing their cell wall compliance. In other aspects, neutral pH can be used to produce yeast with compliant cell walls and/or yeast that is sensitive to cell wall digestive enzymes (eg, glucanase) at all harvest densities. This property is desirable because yeasts with flexible cell walls can exhibit unique immune responses, such as promoting secretion of cytokines (eg, interferon-gamma (IFN-[gamma])) in cells that contain yeast. In addition, these culture methods provide greater accessibility to antigens located in the cell wall. In other aspects, neutral pH is used for certain antigens (such as influenza HA antigens), allowing release by treatment with dithiothreitol (DTT) Disulfide-bound HA, which is not feasible when HA exhibits yeast culture in a lower pH (eg, pH 5) medium. Finally, in other aspects, cells that produce Th1 type cytokines exhibit a yield increase when exposed to the cultured yeast using a neutral pH method (eg, by phagocytosis or other loading). Type cytokines, Th1-type cytokines include, but are not limited to, IFN-γ, interleukin-12 (IL-12), and IL-2.

As used herein, the general use of the term "neutral pH" refers to a pH in the range of between about pH 5.5 and about pH 8, preferably between about pH 6 and about 8. Those skilled in the art will appreciate that there may be minor fluctuations (e.g., one tenth or one percent) when measured by pH. Thus, culturing yeast cells using neutral pH means that the yeast cells are cultured at neutral pH for most of their culture.

In an embodiment of the invention, as an alternative to antigen recombination in a yeast vehicle, the yeast vehicle can be loaded with a protein or peptide antigen or a carbohydrate or other molecule that acts as an antigen in the cell. Subsequently, the yeast vehicle containing the antigen in the cells can be administered to the patient or loaded into a carrier such as dendritic cells (described below). As used herein, a peptide comprises an amino acid sequence of less than or equal to about 30-50 amino acids, while a protein comprises an amino acid sequence of about 30-50 or more amino acids; the protein can be a polymer. A protein or peptide suitable for use as an antigen may be as small as a T cell epitope (ie, greater than 5 amino acids in length) and may be greater than comprising multiple epitopes, protein fragments, full length proteins, chimeric proteins or fusion proteins Any suitable size of the object. Peptides and proteins may be of natural or synthetic origin; such modifications may include, but are not limited to, glycosylation, phosphorylation, acetylation, myristylation, prenylation, palmitoylation, guanylation, and/or addition Glycerol phospholipids inositol. Peptides and proteins can be directly inserted into the yeast of the invention by techniques known to those skilled in the art, such as by diffusion, active delivery, liposome fusion, electroporation, phage, freeze-thaw cycles, and water bath sonication. Fungal media. Yeast vectors that can be directly loaded with peptides, proteins, carbohydrates or other molecules include intact yeasts that can be loaded with antigens after production, but before loading into dendritic cells, as well as protoplast spheroids, 骸 or cytoplasm. Alternatively, the intact yeast can be loaded with an antigen, and then protoplast spheroids, residues, cytoplasts or subcellular particles are prepared therefrom. In this embodiment, the yeast vehicle can carry at least 1, 2, 3, 4 or any number of antigens up to hundreds or thousands of antigens, such as, for example, by loading microorganisms. Provided by the loading of mammalian tumor cells or parts thereof.

In other embodiments of the invention, the antigen is physically attached to the yeast vehicle. Physical attachment of the antigen to the yeast vehicle can be accomplished by any suitable method in the art, including covalent and non-covalent association methods including, but not limited to, antigens such as the use of antibodies or other binding partners The surface is chemically cross-linked to the surface of the yeast vehicle or the antigen is attached to the surface of the yeast vehicle. Chemical crosslinking can be obtained, for example, by a process comprising the treatment of a glutaraldehyde linkage, a photoaffinity label, a carbodiimide treatment, a chemical capable of linking a disulfide bond, and other crosslinking chemical standards in the art. . Alternatively, the chemical can be contacted with a yeast vehicle that alters the charge of the lipid bilayer of the yeast membrane or the composition of the cell wall such that the outer surface of the yeast is more likely to fuse or bind to an antigen having a particular charge characteristic. Targeting agents such as antibodies, binding peptides, soluble receptors, and other ligands can also be incorporated into the antigen as a fusion protein, or otherwise associated with an antigen to allow the antigen to bind to the yeast vehicle.

In yet another embodiment, the yeast vehicle and the antigen are associated with each other by a more passive non-specific or non-covalent bonding mechanism, such as by using the yeast vehicle with the antigen in a buffer or other suitable formulation. Mix gently in the middle. In one embodiment of the invention, both the yeast vehicle and the antigen are loaded intracellularly with a carrier such as dendritic cells or macrophages to form a therapeutic composition or vaccine of the invention. Alternatively, the antigen of the present invention (i.e., the influenza fusion protein of the present invention) can be loaded into dendritic cells in the absence of a yeast vehicle. Various forms in which loading of the two components can be accomplished are discussed in detail below. As used herein, the term "loading" and its derivatives refers to the insertion, introduction, or introduction of components (eg, yeast vehicles and/or antigens) into cells (eg, dendritic cells). Intracellular loading component refers to the intracellular metabolic region in which the component is inserted or introduced into the cell (eg, by mass Membrane and at least loaded into certain intracellular spaces of the cytoplasm, phagosomes, lysosomes or cells). Loading a component into a cell can be referenced (e.g., by electroporation) to force the component into the cell or to place the component in an environment in which the component is substantially accessible to the cell by certain processes, such as phagocytosis ( Any technique such as touching or near cells. Loading techniques include, but are not limited to, diffusion, active delivery, liposome fusion, electroporation, phagocytosis, and water bath sonication. In a preferred embodiment, the dendritic cells are loaded with a yeast vehicle and/or antigen using a passive mechanism comprising phagocytizing the yeast vehicle and/or antigen by dendritic cells.

In one embodiment, the intact yeast (with or without the expression of a heterologous antigen) can be crushed or treated in a manner that produces a yeast cell wall preparation, yeast membrane particles, or yeast fragments (ie, non-intact), and In some embodiments, the yeast fragments can be provided or administered in combination with other compositions including antigens (eg, DNA vaccines, protein subunit vaccines, dead or inactivated pathogens) to enhance the immune response. For example, the yeast can be fragmented into a portion that can be used as an adjuvant using enzymatic treatment, chemical treatment, or physical force (eg, mechanical shear or ultrasonic treatment).

In one embodiment of the invention, the composition or vaccine may also comprise or be capable of producing a biological response modifier compound (i.e., by transfecting a yeast vehicle with a nucleic acid molecule encoding such a modulator) ), although such modulators do not necessarily achieve the strong immune response of the present invention. For example, the yeast vehicle can be transfected or loaded with at least one antigen and at least one biological response modifier compound via at least one antigen and at least one biological response modifier compound, or can be administered in conjunction with at least one biological response modifier Vaccine or composition. Biological response modifiers include compounds that modulate an immune response, which may be referred to as immunomodulatory compounds. Certain biological response modifiers can elicit a protective immune response, while other biological response modifiers can suppress deleterious immune responses. Certain biological response modifiers preferentially enhance cell-mediated immune responses, while other biological response modifiers preferentially enhance humoral immune responses (ie, stimulate immune responses in which cell-mediated immune levels are increased (compared to humoral levels) Reaction, or vice versa). Many techniques for measuring stimulation or inhibition of immune responses and for distinguishing between cell-mediated immune responses and humoral immune responses are known to those skilled in the art.

Suitable biological response modifiers include cytokines, hormones, lipid derivatives, small molecule drugs, and other growth regulators such as, but not limited to, interleukin 2 (IL-2), interleukin 4 (IL-4), White pigment 10 (IL-10), interleukin 12 (IL-12), interferon gamma (IFN-γ) insulin-like growth factor I (IGF-I), transforming growth factor beta (TGF-β) steroids, Prostaglandins and leukotrienes. Yeast vectors can express (ie produce) and possibly secrete IL-2, IL-12 and/or IFN-γ preferentially enhance cell-mediated immunity, while yeast vectors can and may secrete IL-4, IL- 5 and / or IL-10 preferentially enhance humoral immunity. Other suitable biological response modifiers include, but are not limited to, anti-CTLA-4 antibodies (eg, release of unreactive T cells); T cell coactivators (eg, anti-CD137, anti-CD28, anti-CD40); Alemtuzumab (eg CamPath®), denileukin diftitox (eg ONTAK®), anti-CD4, anti-CD25, anti-PD-1, anti-PD-L1, anti-PD- L2 or repression of FOXP3 (e.g., killing CD4 + / CD25 + T regulatory cell activity / kill the cells) agents; of Flt3 ligand, imiquimod ^{(imiquimod) (Aldara TM),} GM-CSF, sargramostim Sargramostim (Leukine®), Toll-like receptor (TLR)-7 agonist or TLR-9 agonist (eg, increasing the number or enhancement of dendritic cells, macrophages, and other specialized antigen presenting cells) An agent that activates dendritic cells, macrophages, and other specialized antigen presenting cells). Such biological response modifiers are well known in the art and are publicly available.

The compositions and therapeutic vaccines of the invention may further comprise any other compound suitable for protecting a subject against a particular disease or condition, including infection by a pathogen, or any compound that can treat or ameliorate any symptoms of the infection.

As used herein, a pharmaceutically acceptable carrier refers to any substance or vehicle suitable for delivery of a yeast vaccine of the invention to a suitable in vivo or ex vivo site. The carrier can include, but is not limited to, an adjuvant, excipient, or any other type of delivery vehicle or carrier.

According to the present invention, adjuvants are generally substances which generally enhance the immune response of an animal to a particular antigen. Suitable adjuvants include, but are not limited to, Freund's adjuvant; other bacterial cell wall components; aluminum-based salts; calcium-based salts; cerium oxide; polynucleotides; toxoids; serum proteins; coat protein; derived from other bacteria of the formulation; interferon gamma]; block copolymer adjuvants, such as Hunter's Titermax adjuvant ^{(CytRx TM, Inc.Norcross, GA)} ; Ribi adjuvants (available from Ribi ImmunoChem Research, Inc. , Hamilton, MT); and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark). The adjuvant of the yeast vaccine of the present invention is not necessarily required, but its use is not excluded.

The carrier is typically a compound that enhances the half-life of the therapeutic composition in the animal being treated. Suitable carriers include, but are not limited to, polymer controlled release formulations, biodegradable implants, liposomes, oils, esters, and glycols.

The compositions and vaccines of the invention may also contain one or more pharmaceutically acceptable excipients. As used herein, a pharmaceutically acceptable excipient refers to any substance suitable for delivering a composition suitable for use in the methods of the invention to a suitable in vivo or ex vivo site. Preferably, the pharmaceutically acceptable excipient is such that the composition is capable of eliciting an immune response at the target site (note that the target can be systemic) after the composition has reached the target cell, target tissue or target in the body. The composition maintains the composition (eg, a yeast vehicle or a dendritic cell containing a yeast vehicle). Suitable excipients of the invention include excipients or formulations (also referred to herein as non-targeting vehicles) that deliver, but do not specifically target, a vaccine. Examples of pharmaceutically acceptable excipients include, but are not limited to, water, physiological saline, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solution, Hank's solution, other physiologically balanced aqueous solutions, oils , esters and diols. The aqueous carrier can contain, for example, suitable auxiliary substances (by enhancing chemical stability and isotonicity) required to approximate the physiological conditions of the recipient. The auxiliary substance may also include a preservative. Stabilizers such as trehalose, glycine, sorbitol, lactose or monosodium glutamate (MSG) may be added to stabilize the vaccine formulation for various conditions, such as temperature changes or lyophilization processes. The composition may also include, for example, sterile water or physiological saline (preferably Buffered suspension.

Yeast vehicles, including those that are administered directly to a patient or first loaded into a carrier such as dendritic cells, can be formulated into the compositions of the present invention using a number of techniques known to those skilled in the art. For example, the yeast vehicle can be dried by lyophilization, which is a preferred embodiment. The yeast vehicle can also be combined with a pharmaceutically acceptable excipient prior to administration with the antigen or loading into dendritic cells or other types of administration. For example, the formulation can be resuspended or diluted in a suitable diluent, such as sterile water, physiological saline, isotonic buffered saline (eg, phosphate buffered to physiological pH), or other suitable Thinner. Lyophilization (freeze drying) is a preferred option. Formulations comprising a yeast vehicle can also be prepared by filling the yeast with a mass or lozenge, such as yeast for baking or brewing operations.

Kit of the present invention

The invention encompasses kits comprising any one or more of the vaccines of the invention and/or any one or more of the Tarmogens or yeast vehicles of the invention, alone or in combination with the antigens and antigenic formulations described herein. The kit of the invention includes any combination of intracellular antigens and extracellular antigens for use in any type of vaccine, and particularly in the yeast vehicle-based vaccine or vaccine strategy of the invention. For example, any of the fusion proteins described herein or protein preparations providing intracellular and extracellular antigens as described herein can be provided in the kit. The antigens may be provided in any form, including: (as Tarmogen) by yeast vehicle delivery or otherwise provided by a yeast vehicle; as a protein preparation (including a fusion protein preparation) or as a dead or inactivated pathogen for DNA In the vaccine. Includes any suitable antigenic form. Multi-antigen or antigen preparations are also included, wherein each antigen is represented by a different yeast vehicle or otherwise provided in a complex, for example, with a different yeast vehicle. For example, various yeast vehicles can exhibit or otherwise provide different antigens from a particular pathogen so that a preferred combination of antigens can be selected for administration to an individual or group of individuals (eg, conservative or internal and/or variable) Or external) and preferred vaccine strategies (eg, conservative antigens or The internal antigen is primed and stimulated by a variable antigen or an external antigen). In one aspect, the kit can additionally include a combination of a Tarmogen for initiating or stimulating a vaccine strategy, either alone or in combination with a kit (as described herein, in a trigger/inspire strategy and similar strategies simultaneously or The antigen preparations are administered sequentially. The kit may also include a yeast vehicle as described herein, which does not exhibit or provide an antigen, and is used as an adjuvant. A set of instructions for use can be included in any of the kits of the present invention. Culture reagents for yeast vehicles can also be included. The yeast cells can be frozen to be cultured or pre-cultured to express the antigen, and then frozen for partial packaging as a kit, or lyophilized.

Method of the invention

One embodiment of the invention is directed to methods of inducing an immune response, including cell-mediated immune responses, humoral immune responses, and combinations thereof. Other embodiments of the invention are directed to methods of protecting an animal against a condition or disease, including infection by a pathogen (e.g., influenza virus infection) or a disease derived therefrom (including prophylactically and/or therapeutically treating a condition or disease). The method comprises the steps of administering a vaccine or composition of the invention as described herein to an animal at or at risk of producing the disease or condition, including a pathogen infection, to slow or prevent the disease or condition, This includes preventing infection or slowing down at least one symptom caused by an infection in the animal. The method of the present invention preferentially induces an antigen-specific cell-mediated immune response against at least one antigen in an animal at least after first administering the vaccine comprising the antigen to the individual. Detailed strategies for customizing an immune response to a particular pathogen or disease and the immune status of the individual to which the vaccine is administered are described above and exemplified herein. Those skilled in the art will be able to conveniently use different combinations of antigens, antigenic expression or type of delivery, and vaccine compositions and vaccine regimens as described herein to achieve the desired immune response.

In the above embodiments, the vaccine or composition comprises: (a) a first yeast vehicle; and (b) any one or more of the antigens described herein (expressed, delivered, secreted, and expressed by the yeast vehicle) Yeast mediation, association and/or mixing). The vaccine may include other yeasts that express, carry, secrete, or mix, associate or complex different antigens as described above. Vehicle. Preferred combinations of antigens expressed, delivered, secreted or mixed, associated or complexed with the yeast vehicle of the present invention, and preferred combinations of yeast vehicles to be combined or sequentially administered by the yeast vehicle of the present invention It has been described in detail above.

In one embodiment, the vaccine comprises at least one antigen that is expressed or provided by the yeast in the cell. In one embodiment, the vaccine comprises at least one antigen that is expressed or provided by the yeast in the cell and at least one antigen that is expressed or provided by the yeast outside the cell. In one aspect of this embodiment, the intracellular antigen is the same antigen as the extracellular antigen, and in other aspects, it is a different antigen. In one embodiment, the antigen expressed or provided by the yeast in the cell comprises at least one antigen that is a conserved antigen or an antigen that is expressed internally by the pathogen. In one embodiment, the antigen expressed or provided extracellularly by the yeast comprises at least one antigen that is a variable antigen or an antigen that is externally (surfaced) by the pathogen. In other embodiments, the variable antigen or external antigen is also expressed or provided by the yeast within the cell.

In one embodiment of the invention, the vaccine comprises at least one antigen expressed or provided by the yeast in the cell and at least one antigen expressed or provided by the yeast outside the cell, and the vaccine is used to elicit a defense ( The immune system of these antigens. Priming means that the vaccine is administered to an individual for the first time such that the individual has not previously been immunized with the antigen or combination of antigens. Thus, the immune system is "primed" to respond more quickly and more efficiently to subsequent encounters with the antigen (eg, by stimulating the vaccine or by encountering with a natural antigen, for example, due to infection with a pathogen).

In one embodiment, the vaccine comprises at least one antigen that is expressed or provided by the yeast in the cell, or at least one antigen that is expressed or provided by the yeast outside the cell, or an antigen that is expressed or provided by the yeast in the cell. The antigen is administered to or provided by the yeast outside the cell, and the vaccine is administered as a challenge vaccine (i.e., after the antigen or the antigen first elicits the first immunization effect).

In one embodiment, where the antigen is highly conserved and unlikely to be mutated, it is possible Need to express a single antigen. In other embodiments, a multi-antigen is required to be expressed or provided than a broader range of cross-protection reactions obtained by targeting a single antigen. Such cross-protective reactions are suitable for the production of universal vaccines which, as described above, can be combined with antibody-producing vaccines to provide a broad range of protective immune responses. Since the protective mechanism is different from the mechanism induced by antibody-producing vaccines, targeted universal antigens can be applied in a dose-saving protocol.

In other embodiments, the elicitor vaccine can be of a different type (eg, a non-yeast based vaccine (eg, protein subunit, DNA, dead/inactivated pathogen) or a different type of yeast vehicle (such as a yeast membrane) A yeast-based vaccine that is either a cell wall particle or a yeast that binds to a protein, DNA, or inactivated/dead pathogen vaccine as an adjuvant. More specifically, in one embodiment of the invention, as described herein The vaccine or composition of the invention may be administered in a regimen comprising administering one or more additional vaccine or immunotherapeutic compositions (including any conventional non-yeast based vaccines or compositions). For example, such Other vaccine or immunotherapeutic compositions may include any other composition comprising an antigen, an antigen encoding or an antigen, such as a DNA vaccine, a dead or inactivated pathogen vaccine or a protein subunit vaccine (eg, a purified antigen preparation). Yeast-based vaccines are preferably used to elicit antigen-specific immune responses, including at least one strong cell-mediated immune response and In the case of a cell-mediated immune response and a humoral immune response, an alternative form of a non-yeast-based vaccine or a yeast-based vaccine is preferably used to elicit an immune response (cell-mediated immune response and/or humoral immune response). Alternatively, there may be instances where the yeast-based vaccine of the invention can provoke an individual's immune response to one or more antigens that have been pre-administered in a non-yeast based vaccine.

In one aspect, a yeast that does not exhibit or otherwise contains or provides a heterologous antigen can be used as an adjuvant along with one or more antigens studied. In one embodiment, a yeast that does not exhibit or otherwise contains or provides a heterologous antigen is used in conjunction with a non-yeast based vaccine (eg, a DNA vaccine) to enhance the immune response of the DNA vaccine. In an alternative, the yeast that exhibits or provides a heterologous antigen (on or in the surface or both) is used in conjunction with other types of vaccines. To enhance the immune response. In other alternatives, yeast that does not exhibit or otherwise contains or provides a heterologous antigen is administered to the individual alone (i.e., does not administer an exogenous antigen). In this aspect of the invention, the individual has carried a sufficient amount of antigen to induce an immune response after administration of the yeast vehicle without the antigen, such as an individual currently infected with the pathogen, an individual undergoing mutation in the cellular protein Or an individual who exhibits or carries an antigen that the immune system does not tolerate or breaks its tolerance.

The heterologous antigen on the yeast vehicle need not be identical to the antigen used in the non-yeast based vaccine in order to elicit a protective immune response. The selection of an antigen allows the two antigens to share sequence similarity, have a common epitope, or can be a different antigen in the target pathogen. In one aspect, an antigen for a yeast-based vaccine and a non-yeast based vaccine is selected to induce a complementary or complementary immune response. This is clearly better compared to the case where the immune response of the heterologous antigen of the yeast vehicle resists the immune response of the antigen in the non-yeast based vaccine.

Preferably, the method of use of a therapeutic composition or vaccine of the invention induces an immune response in an animal such that the animal is protected against or caused by the disease or condition (including infection) Symptoms. As used herein, the phrase "preventing a disease" refers to a reduction in the symptoms of a disease; a reduction in the incidence of the disease and/or a decrease in the severity of the disease. Protecting an animal may refer to a composition of the invention that, when administered to an animal, prevents the onset of the disease and/or treats or slows the symptoms, signs or causes of the disease. Thus, protecting an animal from disease includes preventing the occurrence of a disease (prophylactic or prophylactic vaccine) and treating an animal (a therapeutic or therapeutic vaccine) having the disease or experiencing the initial symptoms of the disease. In particular, protecting animals against disease by inducing an immune response in an animal, by inducing a beneficial or protective immune response (in some cases additionally inhibiting (eg, reducing, inhibiting or repressing) excessively activated or harmful The immune response is completed. The term "disease" refers to any deviation from the normal health of an animal and includes the state when the symptoms of the disease are present, as well as the condition in which the deviation (eg, infection) occurs but the symptoms are still not apparent.

In one embodiment, any of the vaccines of the invention is administered to an individual or group of individuals who have been infected with a pathogen, such as an influenza virus. In other embodiments, any of the vaccines of the invention is administered to an individual or group of individuals at risk of being infected with the pathogen. Such individuals may include a population identified as having a higher risk of influenza infection, for example, than a normal population or a population of individuals. Such individuals may also include a population selected for a particular vaccine of the invention due to the expected pathogen strain (e.g., a viral strain) in the geographic location of the population. These groups can be defined by any suitable parameters. In other embodiments, any of the vaccines of the invention can be administered to any individual or to any group of individuals regardless of their known or predicted state of infection or susceptibility to infection with a particular pathogen.

More specifically, a vaccine as described herein, when administered to an animal by the method of the present invention, preferably produces the following results, including: slowing of infection by the pathogen (eg, at least one symptom of the infection or reduced clinical manifestation), infection Elimination or infection is gradually reduced to eliminate, prevent infection and/or associated symptoms and stimulate effector cell immunity and humoral immunity. In addition, the vaccine preferably elicits the immune system to prevent or reduce all infections of the pathogen, including all life cycle forms, strains or mutants of the pathogen, whether they are free during the cycle or in the cells or tissues of the individual. Preferably, the vaccine also produces a prolonged immunity against the pathogen or at least general or cross-protective immunity to make infection of the new strain or mutant more susceptible to prevention and/or elimination in the future.

The invention encompasses delivery of a vaccine of the composition of the invention to an animal. The administration method can be performed ex vivo or in vivo. Ex vivo administration refers to performing a partial regulatory step outside the patient, such as administering the composition of the invention to a cell removed from a patient (dendritic cells) under conditions such that the yeast vehicle and the antigen are loaded into the cell. ) the group and returning the cells to the patient. The therapeutic composition of the present invention can be administered to a patient or administered to a patient by any suitable mode of administration.

The administration of a vaccine or composition, alone or in combination with a carrier of the invention, is typically systemic or transmucosal. The preferred route of administration is obvious to those skilled in the art. Better Administration methods include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intra-articular administration, intracoronary administration, intra-arterial administration (for example, into the carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration. Administration, intra-articular administration, intraventricular administration, inhalation administration (eg, aerosol), intracranial administration, intraspinal administration, intraocular administration, transaural administration, intranasal administration, oral administration, transpulmonary administration, catheterization, and Inject directly into the organization.

Particularly preferred routes of administration include: intravenous, intraperitoneal, subcutaneous, intradermal, intragangral, intramuscular, transdermal, inhalation, intranasal, oral, intraocular, intraarticular, intracranial, and spinal. Parenteral delivery can include intradermal, intramuscular, intraperitoneal, intrapleural, intrapulmonary, intravenous, subcutaneous, atrial catheter, and venous catheter routes. Transaural delivery can include ear drops, intranasal delivery can include nasal or intranasal injections, and intraocular delivery can include eye drops. Aerosol (inhalation) delivery can also be carried out using standard methods in the art (see, for example, Stribling et al, Proc. Natl. Acad. Sci. USA 189: 11277-11281, 1992, which is incorporated by reference in its entirety. Into this article). For example, in one embodiment, the compositions or vaccines of the invention can be formulated into compositions suitable for spray delivery using a suitable inhalation device or nebulizer. Oral delivery includes solids and liquids which can be used orally and which are suitable for mucosal immunity and which are attributed to compositions containing yeast vehicles which can be readily prepared, for example, as tablets or capsules, and formulated into dietary products for Pass the mouth.

Other routes of administration that regulate mucosal immunity are particularly useful for treating viral infections and other pathogen infections. Such routes include bronchial, intradermal, intramuscular, intranasal, other routes of inhalation, rectal, subcutaneous, topical, transdermal, vaginal and urethral routes.

In one embodiment of any of the above methods, the vaccine is administered to the respiratory tract. In other embodiments, the vaccine is administered by a parenteral route of administration. In still another embodiment, the vaccine further comprises dendritic cells or macrophages, wherein the yeast vehicle or vehicle that expresses the fusion protein (refer to the preferred combination described above) is delivered ex vivo to dendritic cells or macrophages The cells, and dendritic cells or macrophage cell lines containing the yeast agent expressing the antigen, are administered to the animal. In one aspect of this embodiment, the dendritic cells or yeast vehicle have been additionally loaded with free resistance original. In one aspect, the yeast-based vaccine is administered to the same location in the individual as the other yeast-based vaccine or non-yeast based vaccine administered to the individual. In other aspects, the yeast-based vaccine is administered in a different location within the individual than in other yeast-based vaccines or non-yeast based vaccines. In one aspect, the vaccine is administered as a therapeutic vaccine. In other aspects, the vaccine is administered as a prophylactic vaccine.

According to the present invention, an effective administration regimen (i.e., administration of a vaccine or therapeutic composition in an effective manner) comprises inducing an immune response in an animal suffering from a disease or condition or at risk of contracting the disease or condition ( Appropriate dosage parameters and mode of administration are preferred for the animal to be protected against the disease. Effective dosage parameters for a particular disease can be determined using standard methods in this technique. Such methods include, for example, determining survival, side effects (i.e., toxicity), and progression or regression of the disease.

In accordance with the present invention, a suitable single dose size is one that is capable of eliciting an antigen-specific immune response in an animal when administered one or more times over a suitable period of time. The dosage may vary depending on the disease or condition being treated. For example, in one embodiment, a single dose of the yeast vehicle of the present invention is from about 1 x 10 ⁵ to about 5 x 10 ⁷ yeast cells per kg of body weight of the organism to which the composition is administered. equivalent. In a preferred embodiment, each dose of yeast cells does not require adjustment for the body weight of the organism. In this embodiment, the single dose of the yeast vehicle of the present invention is from about 1 x 10 ⁴ to about 1 x 10 ⁹ yeast cells per dose. The amount of yeast vehicle used in a single dose can be between about 0.0001 yeast units (YU) to about 10,000 YU (1 YU = 10 ⁷ yeasts). In one embodiment, the yeast vehicle is used in an amount from about 0.001 YU to about 1000 YU. In other embodiments, the yeast vehicle is used in an amount from about 0.01 YU to about 100 YU. In other embodiments, the yeast vehicle is used in an amount from about 0.1 YU to about 10 YU. In one embodiment, the single dose of the yeast vehicle of the invention is from about 0.1 YU (1 x 10 ⁶ cells) to about 100 YU (1 x 10 ⁹ cells) per dose (i.e., per organism). (Including any intermediate dose in increments of 0.1 x 10 ⁶ cells, i.e., 1.1 x 10 ⁶ , 1.2 x 10 ⁶ , 1.3 x 10 ⁶ ...). This dosage range can be effectively used for any organism of any size, including mice, monkeys, humans, and the like.

When the yeast media by the vaccine agent when administered antigen loaded dendritic cell, preferably a single dose of the vaccine of the present invention for each individual in each administration of about 0.5 × 10 ⁶ to about 40 × 10 ⁶ th Dendritic Cells. Preferably, a single dose per individual of about 1 × 10 ⁶ to about 20 × 10 ⁶ dendritic-cell, and each individual is more preferably from about 1 × 10 ⁶ to about 10 × 10 ⁶ dendritic shape cell.

The "stimulating agent" of the therapeutic composition is preferably administered when the immune response against the antigen is diminished, or when it is desired to provide an immune response against a particular antigen or antigens or to induce a memory response. The elicitor can be administered from about 2 weeks to several years after the initial administration. In one embodiment, the administration process is from about 1 to about 4 administrations of from about 1 x 10 ⁵ to about 5 x 10 ⁷ yeast cells per kg body weight over a period of from about 1 month to about 6 months. Equivalent composition.

In one embodiment of the invention, a dose of the yeast vehicle or vehicle as detailed above is combined with one or more antigens (in one aspect, preferably one dose alone or in combination with one or more The first vaccine of the yeast vehicle or vehicle combined with one or more intracellular antigens of the extracellular antigen is administered to the individual or group of individuals. In the case of influenza, the vaccine preferably comprises at least one or more internal influenza antigens. The vaccine can be administered to an individual within any suitable period of time required to maintain or induce cell-mediated immunity of the defense antigen and to maintain or induce humoral immunity of the defense antigen when the antigen is extracellular. In the context of a pathogen, such as influenza, the vaccine preferably maintains or induces cell-mediated immunity against one or more influenza virus strains. For example, the vaccine can be administered in conjunction with an agonist, or once a year or twice a year, every several years, or otherwise as needed. As described above, this embodiment of the present invention can be used as a general cross-protective vaccine, and can provide immunity against the infection of various types of pathogens longer than conventional vaccines.

In yet another embodiment, a dose of a yeast vehicle or vehicle is included with one or more antigens (the same or different antigens as those included in the first vaccine described above) and preferably comprises a dose alone or together A second vaccine of one or more intracellular antigens of a yeast vehicle or vehicle and one or more extracellular antigens is administered to the same individual or individual receiving the first vaccine described above group. The second vaccine can be administered in conjunction with the first vaccine (eg, as a single vaccine comprising a combination of different yeast vehicles) or separately from the first vaccine. In the latter case, the second vaccine can be administered continuously, but simultaneously with the first vaccine (eg, wherein the dosing interval is seconds, minutes, or hours), or in a different process than the first vaccine, so that Control the immune response induced by various vaccines. For example, if feasible, the first vaccine can be administered once a year or in longer increments with the goal of inducing a general cross-protective cell-mediated immune response and (if applicable based on vaccine design) humoral immune responses . The second vaccine may also be administered once a year or as needed (i.e., once, more than once a year, or in accordance with relevant immunization strategies) to allow the population to immunize against the most prevalent pathogen strains in the population at the appropriate time, or as There is a need to control or prevent infectious diseases or pandemics that are caused by pathogens. This strategy for influenza vaccines is described in greater detail herein, but the invention is not limited to influenza vaccines. As described above, the vaccine strategy of the present invention can be designed to provide cross-protection and pathogen strain/mutant-specific immunity, including cell-mediated and humoral immunity, which is believed to provide a more flexible and effective defense against pathogens than previously described. Immunity. This strategy is, for example, readily applicable to cellular antigens, such as antigens expressed by tumor cells.

In the method of the present invention, vaccines and therapeutic compositions can be administered to animals including any vertebrate (subject, individual, patient), and in particular to any member of the vertebrate or mammalian class. Includes, but is not limited to, primates, rodents, livestock, and domesticated pets. Livestock includes mammals that are to be consumed or produce suitable products (eg, sheep for wool production). Preferred mammals that can be protected include humans, dogs, cats, mice, rats, goats, sheep, cattle, horses, and pigs, and particularly preferably humans.

First and second medical uses

The invention also encompasses the expression or provision of extracellular and/or intracellular antigens (Tarmogens), combinations of extracellular and intracellular antigens, yeast vectors for fusion proteins, yeast vehicles as adjuvants and/or as described herein. Use of any of the described antigenic preparations and/or any combination thereof for the preparation of a formulation or medicament, particularly for the treatment or prevention including pathogen sensation A disease or condition such as infection, cancer, or autoimmune disease. The formulations or drugs can be formulated for any type of administration, including combinations of routes of administration (eg, intranasal and/or parenteral). The formulations or drugs can be prepared for any of the types of administration protocols described herein. In one aspect, the formulation or drug system is used to induce an antigen-specific immune response (cell-mediated and/or humoral immune response), to protect an animal against influenza infection, for treating or preventing a disease or condition For immunization of a population of individuals at risk of infecting a pathogen, such as an influenza virus, for treating a population of infected pathogens, such as influenza viruses, or for protecting animals against infection by pathogens, including influenza viruses.

Influenza composition and vaccine

The use of any of the antigens and the various aspects of the invention described above will be illustrated and exemplified by a detailed discussion of the concepts and embodiments of the present invention for influenza virus use and compositions and methods for inducing an immune response against influenza virus. The invention is not limited to influenza viruses being the source of antigens or antigens.

The inventors of the present invention have developed a yeast-based vaccine comprising a yeast vehicle and one or more influenza virus fusion proteins and methods of use thereof. The yeast vehicle exhibiting one or more influenza antigens or additionally complexed with one or more influenza antigens may be alone or in combination with one or more other one or more other influenza virus fusion proteins or additionally with one or more other influenza virus fusion proteins A combination of yeast agents, or such yeast agents may be in other forms with influenza antigens (including any non-yeast based vaccines (eg, DNA vaccines, protein subunit vaccines, or dead or inactivated influenza viruses) ))combination.

In one embodiment, the vaccine comprises or provides one or more selected from the group consisting of a matrix protein (M1), an ion channel (M2) antigen, a nuclear sheath (NP) antigen, a polymerase PB1 (PB1) antigen, and a polymerase (PB2). A yeast vehicle for the internal influenza antigen in the antigen and polymerase PA (PA) antigen. In other embodiments, the vaccine comprises one or more selected from the group consisting of a hemagglutinin (HA) antigen (any one or more subtypes) and a neuraminidase (NA) antigen (any one or more subtypes) A yeast vehicle for an external influenza antigen. Internal antigen is usually made up of yeast Intracellular performance. External influenza antigens are typically expressed or provided on the surface of the yeast (extracellular antigen) and may also be expressed or provided by the yeast within the cell. In some embodiments, it is preferred to provide both types of antigen (intracellular and extracellular). In a particularly preferred embodiment, the selection may represent an external influenza protein that transmits the most significant virus type or viral population among animal species (eg, humans) during a given time period (eg, over a period of one year), or An external influenza protein that can respond to a potential, suspected or expected specific type of influenza, including an influenza epidemic or a pandemic.

Other and particularly preferred embodiments of the invention are directed to vaccines that utilize a combination of an external influenza antigen and an internal influenza antigen use. In this embodiment, the vaccine comprises expressing or providing at least one member selected from the group consisting of a matrix protein (M1), an ion channel (M2) antigen, a nuclear sheath (NP) antigen, a polymerase PB1 (PB1) antigen, and a polymerase (PB2) antigen. And a yeast vehicle for the internal influenza antigen in the polymerase PA (PA) antigen. The use of such protein combinations is also encompassed by the present invention. The internal influenza antigen is preferably expressed by the yeast in the cell, although the antigen can also be provided extracellularly. The vaccine also includes expressing or providing at least one selected from the group consisting of a hemagglutinin (HA) antigen (any one or more subtypes) and a neuraminidase (NA) antigen (any one or more subtypes) by a yeast vehicle. The external flu antigen in the middle. The external influenza antigen is expressed or provided on the surface of the yeast (extracellular) and can also be expressed or provided by the yeast within the cell. In some embodiments, the performance or provision of both antigenic forms is preferred for external influenza antigens.

Nucleic acid and amino acid sequences of proteins from influenza viruses of various viral strains are known. For example, the sequence of H1N1 of the influenza virus strain A/PR/8/34 is in the NCBI database Accession No. M38279 (the nucleotide sequence is represented herein as SEQ ID NO: 29, which encodes SEQ ID NO: :30) and NC_002019 (nucleotide sequence is represented herein as SEQ ID NO: 31, which encodes SEQ ID NO: 32). For example, the sequence of the avian influenza virus strain A/Vietnam/1203/04 is also known. For example, a nucleotide encoding H5N1 of A/Vietnam/1203/04 (eg, NCBI Library Accession No. AY818135) is represented herein as SEQ ID NO: 33, which encodes SEQ ID NO: 34. . It should be understood that despite this article The specific sequences described are derived from known or reported sequences of such strains, but those skilled in the art can readily select different viral strains or reporter sequences for the same viral strain and can be identical to those described for the disclosed sequences. The method is used in the present invention. It is convenient for us to use any of a variety of sequence software programs to align sequences and to identify sequences of other viral strains or reporter viral sequences corresponding to the sequences of the proteins described herein. In addition, it should be noted that the nucleotide and amino acid sequences may differ slightly in the sequence reports of various strains or public databases. These minor differences are estimated to have no significant effect on the ability to elicit an immune response in accordance with the present invention. The invention is not limited to the sequences described herein. In this embodiment, the yeast vehicle that exhibits or provides an external influenza antigen can be the same or a different yeast vehicle than the yeast vehicle that exhibits or provides an internal influenza antigen. In addition, different combinations of internal influenza antigens and/or external influenza antigens may be expressed or provided on different yeast vehicles, and such media may be used separately or in combination, depending on the desired vaccination. In general, when the influenza antigen is provided by two or more different yeast agents (i.e., as opposed to providing all influenza antigens in a yeast vehicle), the yeast agents can be combined (Mixing) to be administered as a single vaccine (eg, a single injection or other type of administration) or different yeast vehicles can be administered sequentially. Sequential administration can be intervald at any suitable time, including small time increments (seconds or minutes) and longer time increments (day, week, month, or even year). The present invention contemplates that in any of the embodiments, any combination of influenza proteins including at least one internal influenza protein and at least one external influenza protein can be used, and the influenza proteins can be expressed or complexed with the proteins. Any combination of yeast vehicles (including single yeast vehicles) is provided.

There is a great deal of flexibility in how to design and use the vaccine of the present invention. For example, a "general" vaccine comprising a yeast vehicle that provides an internal influenza antigen can be administered to an individual on a regular basis to produce cell-mediated cross-protective immunity in the individual. This vaccine can then be combined, for example, once or periodically with other yeast vehicles that provide an external influenza antigen. Yeast vectors that provide external influenza antigens can be rotated, alternated or selected once a year or based on other Any preferred option (eg, an emergency or expected epidemic or pandemic, or otherwise required) to target the strain of virus of interest and/or the most prevalent strain of virus during a given period of time or for a particular geographic area. Other embodiments of the invention will be apparent from the disclosure provided herein.

In yet another embodiment of the invention, a yeast vehicle that exhibits or provides one or more internal antigens (alone or in combination with one or more external antigens) can be administered as an initiating vaccine, followed by administration of other yeast-based Activators of vaccines or other internal and/or external antigen preparations including, but not limited to, partially purified or purified influenza protein preparations, lysates of yeast vectors expressing influenza proteins, DNA flu vaccine, death ( Or inactivated) a combination of a viral or yeast vehicle (with or without a heterologous antigen) and a non-yeast based vaccine. It should be noted that since the yeast-based vaccine of the present invention is extremely effective in inducing an immune response, an agonist vaccine may not be required, although it is included in an embodiment of the present invention.

Influenza M1, M2 and NP proteins are internal proteins expressed by influenza and exhibit high sequence conservation in influenza virus strains, making them an excellent target for immunotherapy. Administration of the vaccine of the present invention increases influenza-specific CD4+ and CD8+ T cell responses and is expected to reduce viral load and ultimately enhance clearance of the virus in individuals infected with influenza. When the vaccine is combined with an external influenza antigen (for example, HA, M2e (exopeptide of M2 protein) and/or NA antigen) in a yeast-based form, the vaccine can further increase influenza-specific cell-mediated immunity and humoral immunity. To provide a vaccination platform that includes cross-protective vaccination methods (via internal antigens) (with potential long-lasting effects) and strain-specific methods (via external antigens) that are suitable for processing current vaccine needs. Furthermore, the vaccine of the present invention is not based on incubation, making the range of recipients of the vaccine more widely known than the conventional influenza vaccine. Compared with the current flu vaccine, the vaccine is also expected to be more efficient and faster. Finally, as described above, the flexibility in designing the vaccine provided by the present invention is a significant improvement over the conventional influenza vaccine in terms of being able to target the viral subtype as well as to establish general immunity.

One embodiment of the invention relates to a composition (vaccine) useful in a method of protecting an animal from influenza infection or slowing down at least one symptom caused by the influenza infection. The vaccine comprises: (a) a yeast vehicle; and (b) a heterologous influenza fusion protein expressed or provided by the yeast vehicle. As indicated above, the invention encompasses several different influenza fusion proteins that can be used as antigens in the vaccines of the invention. The fusion proteins can be designed to stabilize the expression of the heterologous protein in the yeast vehicle, to prevent post-translational modification of the heterologous protein expressed, and/or in some embodiments to promote the expression of the fusion protein in the yeast vehicle. On the surface. The fusion protein also provides a broad cell-mediated immune response and, in some embodiments, a humoral immune response, and preferably exhibits or provides more than one different influenza antigen, and/or can be associated with other yeasts that exhibit or provide different influenza antigens. A combination of fungicides. Preferably, the combination of antigens comprises at least one internal influenza antigen and at least one external influenza antigen. Although one or more of such fusion proteins can be loaded into a yeast vehicle (eg, a protein) or otherwise complexed or mixed with a yeast vehicle as described herein to form a vaccine of the invention, an embodiment of the invention , but such fusion proteins are most commonly composed of yeast vehicles (eg, whole yeast or yeast protoplast spheroids, which can be further processed as yeast cytoplasts, yeast residues or yeast membrane extracts or Its soluble fraction) is expressed or provided as a recombinant protein.

As mentioned above, the fusion proteins used in the vaccines and compositions of the invention comprise at least one influenza antigen for use in vaccinating animals. The composition or vaccine may include one, two, several, several or a plurality of influenza antigens as desired, including one or more immunogenic domains of one or more influenza antigens. For example, any of the fusion proteins described herein may comprise any one or more selected from the group consisting of influenza matrix protein (M1), influenza ion channel protein (M2) or influenza nuclear sheath protein (NP), and polymerase PB1 (PB1). An internal influenza protein of an antigen, a polymerase (PB2) antigen, and a polymerase PA (PA) antigen, and one or more external influenza proteins selected from the group consisting of influenza hemagglutinin (HA) or influenza neuraminidase (NA) At least part.

According to the invention, the phrase "internal influenza protein" refers to a disease which is manifested by an influenza virus (of any type or strain), wholly or mostly contained in a viral core or in a matrix protein membrane. The protein inside the toxic particles. Among virus types and strains, these proteins are generally highly conserved and can be produced in large quantities by viruses. "External influenza protein" as used herein refers to a protein (eg, a viral surface protein) expressed by an influenza virus (of any type or strain) that runs through the lipid membrane and is mostly expressed on the surface of the viral particle. These proteins can be recognized by antibodies and are therefore suitable for inducing a humoral immune response against the virus. It will be appreciated that influenza ion channel protein (M2), when contained primarily within the influenza virus, has a small extracellular domain (known in the art as M2e) on the surface of the influenza virus. Thus, for the purposes of the present invention, the M2 protein, although generally considered to be an internal influenza protein, is capable of being recognized by an antibody when expressed by an influenza virus or by a cell that exhibits or exhibits at least an extracellular domain on the surface. It can also be considered as an external influenza protein.

In one embodiment of the invention, the influenza antigen portion of the vaccine is produced as a fusion protein comprising two or more antigens. In one aspect, the fusion protein can include two or more immunogenic domains of one or more antigens or two or more antigenic determinants (eg, influenza M1 sequence and influenza HA sequence). The vaccine provides antigen-specific immunity in a wide range of patients. For example, a multidomain fusion protein suitable for use in the present invention can have a plurality of domains, wherein each domain comprises a peptide derived from a particular protein, the peptide comprising at least 4 amino acid residues on both sides and including A mutant amino acid in a protein, wherein the mutation is associated with a particular disease or condition (eg, an influenza infection of a particular strain).

Nucleic acid sequences and amino acid sequences encoding influenza genes and polyproteins from various influenza types, subtypes and strains are known in the art. Thus, those skilled in the art will be able to conveniently produce and use the compositions of the present invention and various influenza-based fusion proteins from influenza strains in vaccines using the guidance provided herein and the reference to specific exemplary influenza antigens.

In the present invention, the present inventors have invented novel recombinant yeast immunotherapy for preventing or inhibiting influenza virus infection. One of the yeast immunotherapy is inductive initiation Influenza matrix protein (M1) that acts as a fusion protein under sub-control. Immunoblot analysis of yeast vaccine cell lysates using anti-histidine-tagged antibodies can demonstrate that recombinant yeasts express the protein. Injection of the M1-presenting yeast vaccine in BALB/c mice induces potent M1 antigen-specific helper T cells and cytotoxic T cell immune responses as demonstrated in lymphocyte proliferation and cytotoxicity assays. Other yeast immunotherapy displays hemagglutinin antigen (HA) protein in cells, and other yeast immunotherapy provides hemagglutinin antigen (HA) protein extracellularly. Some immunotherapies provide antigen under the control of a constitutive promoter. Other yeast vaccines encompassed by the present invention are detailed below.

In one aspect of the invention, the influenza antigen is an internal influenza protein, a matrix protein (M1). In one aspect of the invention, the influenza antigen comprises predominantly from 2 to 252 amino acids of the influenza M1 protein. M1 is an approximately 27 kD influenza protein that forms a matrix protein membrane in influenza virus (see Figure 1). M1 is a structural protein and is involved in viral assembly and ribonucleoprotein (RNP) nuclear export to the cytoplasm. M1 is a highly conserved protein in influenza viruses and is ample viral protein that contains approximately 47% of all viral proteins. Any M1 protein or portion thereof, as well as any mutant or variant of any of these M1 proteins, is contemplated for use in the present invention.

Example 1 describes the preparation of an exemplary vaccine or vaccine component of the invention using a matrix protein (M1 or MP) internal influenza protein (ie, the reference to the component is that the yeast vehicle can be associated with other yeast agents that exhibit different proteins or Further transformation can be performed in combination with other yeast vehicle combinations of other influenza proteins as described herein). In this embodiment, the yeast (eg, S. cerevisiae W303α) can be designed to express an influenza M1 fusion protein derived from the A/PR/8/34 influenza virus under the control of the copper-inducible promoter CUP1. The fusion protein is a single polypeptide having the amino acid sequence SEQ ID NO: 4, which is encoded by the nucleic acid sequence designated SEQ ID NO: 3.

In other aspects of the invention, the influenza antigen is an external influenza protein, hemagglutinin (HA). In one aspect of the invention, the influenza antigen mainly comprises influenza HA protein 2 to 530 amino acids, including the N-terminal ER target signal sequence of HA, but excluding the 36 C-terminal residues of HA, thereby removing its C-terminal membrane anchor and cytoplasmic tail. In other aspects, the influenza antigen mainly comprises 17 to 342 amino acids of the influenza HA protein, which does not include the N-terminal ER target signal sequence of the 16 amino acids of HA, but does not include the C-terminus of HA. Membrane anchors and 36 C-terminal residues of the cytoplasmic tail. HA is a membrane intrinsic protein expressed on the surface of influenza virus particles (see Figure 1). The hemagglutinin causes the host cell to bind to the surface of the target cell via the citrate residue of the glycosylation receptor protein, and after the virus is contained by endocytosis, the viral membrane and the host membrane are subsequently in the endosome Fusion. HA contains at least four primary antigenic sites, and replacement of only monoamine acids in one of the four regions results in the virus escaping immune surveillance and spreading worldwide each year. Three different HA proteins have been identified in human infections, known as H1, H2, and H3; 13 other species have been found in animal influenza viruses, including H5 found in avian influenza viruses. Any HA protein or portion thereof, as well as any mutant or variant of any of these HA proteins, is contemplated for use in the present invention. In one embodiment, the yeast vehicle of the invention exhibiting a HA protein exhibits more than one HA protein (eg, H1, H2, etc.) or is combined with a yeast vehicle that exhibits a different HA protein (eg, exhibiting H1 A vehicle and a vehicle that exhibits H2 or other HA protein are included in the vaccine.

Example 2 describes the preparation of other exemplary vaccines or vaccines of the invention using hemagglutinin (HA) external influenza proteins. In this embodiment, the yeast (eg, S. cerevisiae W303α) is designed to express the fusion protein under the control of the TEF2 promoter. A fusion protein comprising an influenza HA antigen (H1) derived from an A/PR/8/34 influenza virus is a single polypeptide comprising the amino acid sequence SEQ ID NO: 6, which is represented by SEQ ID NO: 5 herein. The nucleic acid sequence is encoded.

Example 2 also describes the use of other hemagglutinin (HA) external influenza proteins (in this case, H5 HA from avian influenza virus strains) to prepare other exemplary vaccines or vaccine components of the invention. In this embodiment, the yeast (eg, S. cerevisiae W303α) is designed to represent a fusion protein. Contains influenza HA antigen derived from A/Vietnam/1203/04 influenza virus The fusion protein of (H5) is a single polypeptide comprising the amino acid sequence SEQ ID NO: 20, which is encoded by the nucleic acid sequence represented by SEQ ID NO: 19 herein.

The above two fusion proteins can be designed to express HA fusion proteins in cells by yeast. Briefly, the fusion proteins contain the N-terminal signal sequence of Aga2 and HA. The signal sequence of Aga2 is targeted for fusion to translocate into the ER, but the signal sequence of HA acts as a terminator. Therefore, the fusion protein cannot pass through the secretory pathway. It becomes a membrane intrinsic protein whose HA moiety remains on the inner side of the plasma membrane.

Example 3 describes the preparation of a further exemplary vaccine or vaccine component of the invention using hemagglutinin (HA) external influenza protein. In this embodiment, the yeast (eg, S. cerevisiae W303α) is designed to express the fusion protein under the control of the TEF2 promoter. This fusion protein is designed to provide an N-terminal portion of the HA (H1) antigen (referred to as HA1) derived from the A/PR/8/34 influenza virus by the yeast. This protein can be localized to the outside of the cell wall of the yeast cell when it is expressed in cells which also express Aga1p, but can also be contained in the cell. The fusion protein comprises the amino acid sequence SEQ ID NO: 10, which is encoded by the nucleic acid sequence represented by SEQ ID NO: 9 herein.

In other aspects of the invention, the influenza antigen is an external influenza protein, a neuraminidase (NA), and in other embodiments, the immunogenic portion of NA is contemplated. NA is a membrane intrinsic protein expressed on the surface of influenza virus particles (see Figure 1). The neuraminidase digests the citrate (neuraminic acid) found on the surface of most cells. Since tannic acid is part of the viral receptor, when the virus binds to the cell, it is internalized (endocytosis). At the end of the infection, niacin has been removed from the surface of the infected cells by the neuraminidase, making it easier for the progeny virions to spread out once they leave the cell. The neuraminidase is also involved in penetrating the mucus layer in the respiratory tract. Two different NA proteins have been discovered in human infections, called N1 and N2; the other seven have been found in animal influenza viruses. Any NA protein or portion thereof, as well as any mutant or variant of any of these NA proteins, is contemplated for use in the present invention. In a preferred embodiment, the yeast vehicle of the invention exhibiting a NA protein exhibits more than one NA The protein (eg, N1, N2, etc.), or in combination with a yeast vehicle that exhibits a different NA protein (eg, one that exhibits NA and one that exhibits NA or one of the other NA proteins) is in the vaccine.

In still another aspect of the present invention, the influenza antigen used in the vaccine of the present invention is an internal or external influenza protein, ion channel protein (M2). In one aspect of the invention, the influenza antigen comprises predominantly the extracellular portion of the influenza M2 protein, also known as M2e. In one aspect, M2e is fused to the C-terminus of the NP influenza protein or a portion thereof. This protein (M2e) can be designed to be intracellular (intracellular). In other embodiments, M2e is fused to a cell wall protein (eg, Aga2) for expression on the extracellular surface of the yeast. M2 is a matrix protein and is a membrane intrinsic protein that spans the matrix protein membrane and the lipid bilayer and is expressed on the surface of the virus particles (see Figure 1). M2 is an ion channel that allows protons to enter the viral particle during detachment of the virion within the endosome, and it also modulates the pH of the trans-Golgi network in the virus-infected cells. M2 is a homo-oligomer of 97 residues to a single transmembrane (TM) domain whose residues include the pore regions of the channel. The biologically active form of the channel is a homotetramer. According to the invention, the portion of the M2 protein that is externally expressed on the virus (i.e., also known as the extracellular domain of M2) may be referred to herein as M2e. M2e is known to be highly conserved among influenza A viruses. As described above, in one embodiment of the invention, a portion of the M2 protein comprising predominantly or exclusively comprising M2e is expressed by a yeast vehicle. In this embodiment, the M2 protein is considered an external influenza protein. In other embodiments, the M2 protein can be considered an internal influenza protein when representing a portion of the matrix protein membrane of the M2 protein. Any M2 protein or portion thereof, as well as any mutant or variant of any of these M2 proteins, is contemplated for use in the present invention.

In other aspects of the invention, the influenza antigen used in the vaccine of the invention is an internal influenza protein, a nuclear sheath protein (NP) (also known as a nuclear protein). In one aspect of the invention, the influenza antigen comprises predominantly a portion of the influenza NP protein that is expressed in the cell fluid of the yeast and is an immunogen. NP is a protein localized in a shell formed by a matrix protein membrane (see Figure 1). The main function of NP is to encapsidize the viral genome to form RNA transcription, Replicating and encapsulating ribonucleoprotein (RNP), but NP can perform other essential functions throughout the viral life cycle. According to NP, influenza can be classified into type A, type B and type C, but NP is highly conserved even between each virus type and especially between type A and type B. Any NP protein or portion thereof, as well as any mutant or variant of any of these NP proteins, is contemplated for use in the present invention. In a preferred embodiment, the yeast vehicle of the invention exhibiting an NP protein exhibits more than one NP protein (eg, NP from influenza A and NP from influenza B), or is associated with a different NP protein. A yeast vehicle combination (eg, a vehicle that exhibits an NP from influenza A and a vehicle that exhibits an NP from influenza B) is in the vaccine.

Example 4 describes the preparation of a further exemplary vaccine or vaccine component of the invention using hemagglutinin (HA) external influenza protein. In this embodiment, the yeast (eg, Saccharomyces cerevisiae) is designed to express the fusion protein under the control of the TEF2 promoter. This fusion protein is designed to provide an HA (H5) antigen derived from the A/Vietnam/1203/04 influenza virus strain which is expressed extracellularly by yeast. This protein can be localized to the outside of the cell wall of the yeast cell when it is expressed in cells that also exhibit Aga1p. The fusion protein comprises the amino acid sequence SEQ ID NO: 14, which is encoded by the nucleic acid sequence represented by SEQ ID NO: 13 herein.

Example 5 describes several various structures that have been expressed by the yeast vehicle of the present invention and which are described for surface expression (including yeast protoplast spheroids) and additionally illustrate the effect of glycosylation on surface expression, HA-containing fusion proteins. design.

One fusion protein, designated TK75-15, is a fusion protein indicated herein as SEQ ID NO: 36, which is encoded by SEQ ID NO: 35 and which illustrates the fusion protein structure of the HA sequence at the C-terminus of the Aga2 sequence. This protein, when expressed in a yeast cell that also expresses Aga1p (in this case, driven by the CUP1 promoter), is localized to the outside of the cell wall of the yeast cell, as well as in the cell fluid, as shown in Figure 10B (top left). .

Example 5 also depicts a fusion protein (denoted VK4), schematically shown in Figure 10B (upper right), designed to localize influenza HA protein (Hl) to the cell wall (driven by the TEF2 promoter) using the Aga2 sequence. In this structure, the protein is constructed to form the HA sequence at the N-terminus of the Aga2 sequence. When the performance of this protein in native Agalp also performed (in this condition, its native promoter) Mat a positioning of the yeast cell wall at the outside of yeast cells, Qieyi present in the cell. The fusion protein comprises the amino acid sequence SEQ ID NO: 26, which is encoded by the nucleic acid sequence represented by SEQ ID NO: 25 herein.

Example 5 also depicts a fusion protein similar to VK4 above (denoted VK11) except that the fusion protein was designed to localize the influenza H5 HA protein to the cell wall (driven by the TEF2 promoter) using the Aga2 sequence. In this structure, the protein is also constructed to form the HA sequence at the N-terminus of the Aga2 sequence. The fusion protein comprises the amino acid sequence SEQ ID NO: 22, which is encoded by the nucleic acid sequence represented herein as SEQ ID NO:21.

Example 5 also depicts a fusion protein (denoted VK8), schematically shown in Figure 10B (bottom left), designed to localize influenza HA (Hl) protein to the cell wall (driven by the TEF2 promoter) using the Cwp2 sequence. In this structure, the protein is also constructed to form the HA sequence at the N-terminus of the Cwp2 sequence. This protein can be localized to the outside of the cell wall of the yeast cell and can also be present in the cell. The fusion protein comprises the amino acid sequence SEQ ID NO: 28, which is encoded by the nucleic acid sequence represented by SEQ ID NO: 27 herein.

Example 5 also depicts a fusion protein similar to VK8 above (denoted as VK12) except that the fusion protein was designed to localize the influenza H5 HA protein to the cell wall (driven by the TEF2 promoter) using the Cwp2 sequence. In this structure, the protein is also constructed to form the HA sequence at the N-terminus of the Cwp2 sequence. The fusion protein comprises the amino acid sequence SEQ ID NO: 24, which is encoded by the nucleic acid sequence represented by SEQ ID NO: 23 herein.

Example 5 also depicts a fusion protein (denoted as Lu002), schematically shown in Figure 10B (bottom right), designed to have an endogenous alpha-factor signal and a leader sequence to express influenza HA protein (driven by the TEF2 promoter), The influenza HA protein has the intact transmembrane domain in the plasma membrane of the yeast protoplast spheroid. This protein (the alpha-factor signal and the leader sequence naturally cleave from the protein in Golgi) is localized to the plasma membrane of the yeast protoplast spheroid and may also be present in the cell. Introduction of foreign proteins into yeast using endogenous alpha-factor signaling and leader sequences The bacterial secretion channel has been previously described (for example, see U.S. Patent No. 5,413,914; or Franzusoff et al., J. Biol. Chem. 270, 3154-3159 (1995)).

Example 6 describes the preparation of a further exemplary Tarmogen or Tarmogen component of the invention using several internal influenza proteins. In this embodiment, the yeast (eg, Saccharomyces cerevisiae) is designed to express the fusion protein under the control of the TEF2 promoter. The fusion protein is designed to provide M1 antigen and NP antigen (derived from A/PR/8/34 influenza virus) and M2e antigen (including A/PR/8/34 influenza virus strain and A from the yeast) in the cell. /Vietnam/1203/04 influenza virus strain M2e antigen). The yeast-based vaccine of the present invention is suitable for inducing cross-protective immunity against antigens that are conserved among all influenza virus strains. The fusion protein comprises the amino acid sequence SEQ ID NO: 16, which is encoded by the nucleic acid sequence represented by SEQ ID NO: 15 herein.

Example 7 describes the preparation of a further exemplary Tarmogen or Tarmogen component of the invention using several internal influenza proteins. In this embodiment, the yeast (eg, Saccharomyces cerevisiae) is designed to express the fusion protein under the control of the TEF2 promoter. The fusion protein is designed to provide intracellular representation of the NP antigen derived from the A/PR/8/34 influenza virus and the M2e antigen derived from the A/PR/8/34 influenza virus. The yeast-based vaccine of the present invention is suitable for inducing cross-protective immunity against antigens that are conserved among all influenza virus strains. The fusion protein comprises the amino acid sequence SEQ ID NO: 18, which is encoded by the nucleic acid sequence represented by SEQ ID NO: 17 herein.

In one embodiment of the invention, any one of the above influenza virus antigens is expressed together with at least one other influenza virus antigen in the yeast vehicle of the invention. Preferably, the internal influenza antigen (eg, M1, M2 or NP) is expressed together with an external influenza antigen (eg, HA, NA or M2e). These influenza antigens can be expressed using the same or different structures. The external influenza antigen can be provided extracellularly by the yeast and/or provided intracellularly. Preferred combinations of antigens to be expressed by yeast vehicles include, but are not limited to, M1 and HA; M1 and NA; M1, HA and NA; NP and HA; NP and NA; NP, HA and NA; HA; M2 And NA; M2 and M1; M2, HA and NA; and M1, M2 and NA. In any of the combinations including M2, M2 can be any portion of full length M2 or M2e or M2 that can be expressed or provided in yeast and is immunogenic. Similarly, other proteins can be expressed in any form, portion or variant described herein. In such combinations, any one or more of the subtypes of the proteins can be expressed or provided, and in particular, any one or more of the HA or NA subtypes can be expressed or provided.

In another embodiment of the invention, any of the above-described yeast agents that exhibit or provide one or more influenza antigens or a combination of such antigens can be associated with yeast that exhibits or provides one or more different antigens or combinations of such antigens. The bacterial vehicles are combined to form a yeast vaccine. Alternatively, any of the above-described yeast vehicles that exhibit or provide one or more influenza antigens can be administered sequentially with a yeast vehicle that exhibits one or more different antigens or a combination of such antigens. Preferably, the yeast vehicle that exhibits or provides one or more internal influenza antigens can be administered in combination or sequentially with a yeast vehicle that exhibits or provides one or more external influenza antigens. Preferred combinations of yeast vehicles include, but are not limited to, the performance of co- or sequential administration of a yeast vehicle that exhibits or provides HA, NA, or a combination thereof, including one or more subtypes of such antigens, or A yeast vehicle that provides M1; a yeast vehicle that exhibits or provides a NP with a yeast agent that exhibits or provides HA, NA, or a combination thereof (including one or more of the antigens) And a yeast vehicle that provides a co- or sequential administration of a yeast vehicle that exhibits or provides HA, NA, or a combination thereof (including one or more subtypes of the antigens) or provides M2. In another embodiment, a yeast vehicle that exhibits or provides any two or more of M1, NP, and/or M2 can be expressed or provided with HA, NA, or a combination thereof (including one of the antigens or A variety of subtypes of yeast vehicles are administered together or sequentially.

Isolation of fusion proteins, nucleic acid molecules and cells

Another embodiment of the invention encompasses an isolated protein comprising any of the isolated fusion proteins comprising an influenza antigen as described herein. The invention also includes isolated nucleic acid molecules encoding any of the proteins, recombinant nucleic acid fragments comprising the nucleic acid sequences encoding the proteins And cells and vectors comprising or transfected/transformed with or by the nucleic acid molecules or recombinant nucleic acid molecules, including viral vectors.

Preferred fusion proteins of the invention include any of the fusion proteins described herein. Exemplary fusion proteins encompassed by the invention include fusion proteins comprising or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO : SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 36 . Since various influenza protein sequences are well known in the art, other fusion protein sequences are readily apparent to those skilled in the art, in conjunction with the teachings provided herein.

The invention also includes any nucleic acid molecule comprising or consisting essentially of a nucleic acid sequence encoding any of the fusion proteins described herein.

Suitable host cells transfected with a recombinant nucleic acid molecule of the invention include any cell that can be transfected or transformed, including any animal, insect, bacterial, fungal (including yeast) cell. In one embodiment, the host cell is an animal cell that has been transfected with a fusion protein of the invention and that exhibits the protein. Such cells are illustrated in the Examples section and are, for example, suitable for assessing antigen-specific T cell responses induced by the vaccines or compositions of the invention. Other transfected cells can also be tested for other vaccines or compositions against influenza antigens.

The results of the following experiments are provided for illustrative purposes and are not intended to limit the scope of the invention.

Instance Example 1

The following example describes the design of the influenza M1 fusion protein yeast vaccine GI-8001 of the present invention (also commonly referred to as GI-8000 in some of the figures).

S. cerevisiae is designed to express influenza M1 fusion protein under the control of the copper-inducible promoter CUP1. A fusion protein is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID) NO: 4): 1) sequence preventing MDE degradation (positions 1 to 6 of SEQ ID NO: 4) MADEAP (SEQ ID NO: 1); 2) amino acid 2 to 252 of M1 protein (SEQ ID NO: 4 positions 7 to 257); 3) triglycine spacers which are introduced to separate the M1 protein from the histidine label (positions 258 to 260 of SEQ ID NO: 4); and 4) C-terminal hexamer Histidine label (positions 261 to 266 of SEQ ID NO: 4). The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 2 is represented herein as SEQ ID NO: 1. In this exemplary yeast-based vaccine, the M1 gene is selected by RT-PCR in cultured cells infected with influenza A/PR/8/34/H1N1, and the encoded amino acid sequence and the virus are encoded. The theoretical M1 amino acid sequence of the strain/genotype is perfectly matched.

The yeast (Saccharomyces cerevisiae W303) expressing the influenza M1 fusion protein was cultured and induced in ULDM medium. The pH of this medium was not adjusted to the neutral pH conditions as described herein (i.e., using normal yeast culture conditions). The performance of the influenza M1 fusion protein was induced with 0.375 mM copper sulfate at 0.2 YU/ml. Yeasts are harvested at the mid-index stage and heat killed. The performance of the M1 fusion protein was confirmed by Western blot analysis of the lysate from the copper-induced heat inactivation of GI-8001 yeast (see Figure 2). Protein detection uses a monoclonal antibody-specific histidine label.

The following are standard formulations for ULDM media:

Female BALB/c mice were injected once a week with 3 doses of PBS alone or 1YU or 10YU A GI-8001 yeast-based vaccine showing M1 (shown as "1YU M1" and "10YU M1" in Figures 3A and 3B, respectively). Mice died 16 days after receiving the final dose. In vitro stimulation assays were performed using yeast expressing M1 as a target (IVS-M1) or using attenuated influenza virus as a target (IVS-flu) to evaluate CTL response and lymphocyte proliferation, and collecting supernatants The solution is used for cytokine analysis. P815 tumor cells, as target cells in these assays, are transfected with influenza M1 fusion protein or infected with influenza. The results of the CTL assay are shown in Figure 3. These results demonstrate that vaccination of mice with GI-8001 induces antigen-specific (M1 and influenza virus) CTL responses.

Figure 4 shows Western blot analysis of lysates from P815 cells infected with influenza A/PR/8/34. HA was identified in the supernatant, indicating that P815 cells can be infected with influenza virus and can be used as target cells in assays described herein.

Figures 5 and 6 show the results of lymphocyte proliferation assays. In Fig. 5, the stimulating antigen is a lysate of a yeast (MP yeast) expressing an M1 fusion protein. In Figure 6, the elicited antigen is death flu (A/PR8). These results demonstrate that GI-8001 (GI-8000, M1-presenting yeast) induces a yeast-specific proliferative response.

Example 2

The following examples describe the design of two yeast-based vaccines that express hemagglutinin (HA) in the cells of the present invention, the first using H1 HA (also referred to elsewhere as GI-8002 or GI-8000-I herein). And the second uses H5 HA (also known as GI-8102).

H1-HA for intracellular expression

S. cerevisiae is designed to express HA (H1 or HA1) fusion proteins under the control of the transcription elongation factor 2 promoter TEF2 (see Figure 7 below). A fusion protein comprising an influenza HA antigen is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID NO: 6): 1) Full length Saccharomyces cerevisiae Aga2 protein sequence (positions 1 to 87 of SEQ ID NO: 6), including its native 18 amino acid ER-target signal sequence (positions 1 to 18 of SEQ ID NO: 6); 2) influenza HA protein Amino acid 2 to 530 (positions 88 to 616 of SEQ ID NO: 6), which comprise the N-terminal ER-target signal sequence of HA (positions 88 to 105 of SEQ ID NO: 6), but excluding 36 C- of HA a terminal residue to remove its C-terminal membrane anchor and cytoplasmic tail; 3) a triglycine spacer that separates the HA protein bulk from the histidine label (positions 617 to 619 of SEQ ID NO: 6); C-terminal hexahistidine labeling (positions 620 to 625 of SEQ ID NO: 6). The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 6 is represented herein as SEQ ID NO:5. This fusion protein and the Tarmogen expressing the protein may be referred to as GI-8000 Aga2-HA or GI-8002.

In the exemplary yeast-based vaccine, the HA gene line is selected from cultured cells infected with influenza A/PR/8/34/H1N1, and the encoded amino acid sequence and the virus strain/genotype are The theoretical HA amino acid sequence does not match exactly. The alignment of the actual sequence with the theoretical sequence is shown in Table 1 (see Example 2).

The yeast containing TEF2::Aga2-HA was cultured in UDM to a medium index stage. Using this medium, it is not necessary to adjust the culture conditions to neutral pH conditions. The cells are washed and heat killed and all proteins are extracted from the cells. HA fusion protein performance by west The dot blot method was used to confirm the lysate from the heat-killed yeast expressing the protein (Fig. 7). Western blot analysis of all proteins detected by his-tagged mAb (right of Figure 7) or HA-specific mAb (not shown) indicates that the Aga2-HA protein accumulates to high levels in the yeast.

The following are standard formulations for ULDM media:

BALB/c female mice (5-10 weeks old) were administered PBS, 0.5YU GI-8000 Aga2-HA (GI-8002) by subcutaneous (100 μl) or intranasal (50 μL) once a week for three weeks. 5YU GI-8000 Aga2-HA (GI-8002). The mice died two weeks after the third dose. Serum was collected for analysis and an in vitro stimulation assay was performed on splenocytes to evaluate CTL responses (using ⁵¹ Cr-labeled homologous P815 tumor cells that had been infected overnight with A/PR/8/34 influenza virus) and lymphocyte proliferation. For the proliferation assay, spleen cells were cultured for five days with GI-8000 Aga2-HA (GI-8002) or UV-inactivated A/PR8 influenza virus. Serum was also collected for antibody analysis.

Figure 8 shows the results of the CTL assay, and Figure 9 shows the results of the lymphocyte proliferation assay. These results demonstrate that GI-8000 Aga2-HA can induce influenza virus-specific CTL responses. Administration by subcutaneous route generally induces a stronger CTL response than intranasal administration. However, low dose intranasal administration can induce high levels of CTL response. Proliferation assays demonstrate that two routes of administration induce influenza virus-specific lymphocyte proliferation, despite intranasal routes of administration and subcutaneous administration A lower level of proliferative response can be produced.

The above-described Tarmogen, which expresses HA (as an intracellular antigen), was also evaluated for its ability to induce a humoral response as an adjuvant. In particular, two doses of antigen (from A/) were administered on days 0 and 21 in the presence or absence of adjuvant (adjuvant as GI-8000-I Tarmogen as described in this example) or Alum. HA of PR/8/34). The HA neutralizing antibody titer was measured for 3 weeks after the second dose of antigen. Table 2 shows the results of this experiment. This data demonstrates that GI-8000-I has excellent ability to act as an adjuvant for B cell responses. In fact, with or without the addition of alum as an adjuvant, the effect is equally good or exceeds the purified protein. Thus, the same efficacy (i.e., dose saving) for producing an antibody response can be obtained using a lower dose of a non-yeast based vaccine.

The above-described Tarmogen, which expresses HA (as an intracellular antigen), was also evaluated for its ability to elicit an immune response against influenza. Priming with sufficient virions of inactivated A/PR/8/34 to produce 10 μg of HA. Use 5YU GI-8000-I. Give it a boost after about 1 month. After one month, the HI (HA Neutralization Inhibition) titer was measured. Excitation is performed while measuring the HI titer. The virus titer was measured after 5 days. Table 3 shows the results when using GI-8000-I to trigger defense against influenza.

Using this type of Tarmogen, T cells that protect against multiple antigens are produced in influenza-infected cells. The benefit of inducing an immune response against multiple antigens is that it allows cross-protective responses and allows multiple antigens exhibited by Tarmogen to act as a general vaccine. This type of vaccine can be dose-saving (i.e., the dose required for efficacy is lower) by generating an effective immune response compared to conventional vaccines for avian or seasonal influenza.

H5 HA for intracellular expression

S. cerevisiae is designed to express a HA (H5) fusion protein (also referred to herein as GI8102) in cells. A fusion protein comprising an influenza H5 antigen is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID NO: 20): 1) Full length Saccharomyces cerevisiae Aga2 protein sequence (positions 1 to 87 of SEQ ID NO: 20), including its 18 native amino acid ER-target signal sequences (positions 1 to 18 of SEQ ID NO: 20); 2) equivalent to H1 N-terminal ER target signal sequence of 1 to 16 residues of HA (positions 88 to 105 of SEQ ID NO: 20); 3) H5 HA of avian influenza virus strain A/Vietnam/1203/2004 (SEQ ID NO: Position 20 to 106); it does not include 36 C-terminal residues of HA, thereby removing its C-terminal membrane anchor and cytoplasmic tail; And 4) C-terminal hexahistidine labeling (positions 621 to 626 of SEQ ID NO: 20). The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 20 is represented herein as SEQ ID NO: 19.

Example 3

The following examples describe the design of another HA fusion protein (referred to herein as HA1) of the yeast vaccine of the invention (which is generally referred to herein as GI-8000-S).

This fusion protein is designed to provide extracellular expression by yeast as well as intracellular expression of HA fusion proteins. A fusion protein comprising an N-terminal portion of influenza HA antigen (HA1) is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID NO: 10): 1) full-length S. cerevisiae Aga2 protein sequence (positions 1 to 89 of SEQ ID NO: 10), including its 18 natural amino acid ER-target signal sequences (positions 1 to SEQ ID NO: 10) 18); 2) Amino acids 17 to 342 of influenza HA protein (positions 90 to 415 of SEQ ID NO: 10), which do not include the 16th amino acid N-terminal ER-target signal sequence of HA and do not include C-terminal membrane anchor and 36 C-terminal residues of HA at the cytoplasmic tail; 3) Triglycine spacer separating HA1 protein bulk from histidine label (positions 416 to 418 of SEQ ID NO: 10) And 4) C-terminal hexahistidine labeling (positions 419 to 424 of SEQ ID NO: 10). This protein localizes to the outside of the cell wall of the yeast cell when it is expressed in cells that also exhibit Aga1p (see Figure 10A, which schematically depicts the extracellular appearance using this technique). The protein can also be expressed in cells. The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 10 is represented herein as SEQ ID NO:9.

In this exemplary yeast-based vaccine, the HA gene line is housed in the influenza A/PR/8/34/H1N1 egg bank, and the encoded amino acid sequence and the theoretical HA amine group of the virus strain/genotype The acid sequence does not match exactly. The alignment of the actual sequence with the theoretical sequence is shown in Table 4. There are 10 mismatched amino acids between the two sequences.

Table 4: Alignment of the theoretical HA1 region (SEQ ID NO: 11) with the actual HA1 sequence (SEQ ID NO: 12) cloned herein.

The expression of the fusion protein was confirmed by Western blot analysis of copper-induced heat-inactivated yeast lysates (see Figure 17). After normal (non-neutral pH) conditions, induced by copper in UDM (to induce Aga1 synthesis), the constitutively expressed Aga2-HA1 was detached from Aga1 at the cell surface of living cells with 50 mM DTT, and DTT was dissolved. The solution was treated with glycosidase PNGase F or ENDO-h for 1 h. The Aga-HA1 content of the reactants was analyzed by Western blotting (Fig. 17). The intracellular and cell surface HA1 expression was observed.

Example 4

The following example describes the design of another HA fusion protein yeast vaccine of the present invention.

Another yeast vehicle designed to express hemagglutinin (HA) protein (also known as TK88) from avian influenza virus strain A/Vietnam/1203/2004 (H5-N1) as a yeast cell surface (extracellular) protein (Aga2-H5 HA)), which can also be localized in cells. Referring again to the top view in Fig. 10A, the structure for surface representation is illustrated. Contains flu The fusion protein of HA antigen (H5) is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID NO: 14): 1) Full-length Saccharomyces cerevisiae Aga2 protein sequence (amino acid 1 to 87 of SEQ ID NO: 14), including its natural 18th amino acid ER-target signal sequence; 2) Amino acid 2 to 530 of influenza H5 HA protein ( Positions 88 to 616) of SEQ ID NO: 14 lacking the N-terminal ER-target signal sequence of HA and also excluding 36 C-terminal residues of HA, thereby removing its C-terminal membrane anchor and cytoplasmic tail; 3) a triglycine spacer separating the HA protein bulk from the histidine label (positions 617 to 619 of SEQ ID NO: 14); and 4) a C-terminal hexahistidine label (SEQ ID NO: 14) Positions 620 to 625). This protein is localized to the outside of the cell wall of the yeast cell as well as the cytosol and ER when expressed in cells that also express Aga1p. The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 14 is represented herein as SEQ ID NO: 13.

This Tarmogen can be expressed using the neutral pH conditions described herein and under normal yeast culture conditions. When expressed using neutral pH conditions, the fusion protein is detected on the cell wall (surface). Neutral pH conditions increase the efficiency of protein expression and improve the ability of proteins present on the surface due to the effect of pH on the cell wall and the ability of HA-specific antibodies to detect proteins on the surface.

Example 5

The following examples describe the production of other constructs (extracellular constructs) in which the antigen is designed to target the surface of the yeast.

Other Tarmogens are designed to express the hemagglutinin (HA) protein from influenza as a yeast cell surface (extracellular) protein. The protein can also be expressed in yeast cells. Referring again to the top view in Fig. 10A, the structure for surface representation is illustrated.

Figure 10B schematically illustrates several specific configurations for surface representation of antigens and shows how to use various yeast proteins as spacer arms. While such constructs can be used to express influenza HA, any protein can be expressed using such methods and constructs.

Aga2-HA H1 fusion protein (surface)

The fusion protein (shown as TK75-15), schematically shown in Figure 10B (top left), was designed to express the influenza HA protein on the cell wall (driven by the TEF2 promoter) using the Aga2 sequence. In this construction, the protein is constructed to form the HA sequence at the C-terminus of the Aga2 sequence. This protein, when expressed in cells that also exhibit Aga1p (in this case, driven by the CUP1 promoter), is localized to the outside of the cell wall of the yeast cells as well as to the cytosol as shown in Figure 10B (top left). A fusion protein comprising an influenza HA antigen is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID NO: 36): 1) Full length Saccharomyces cerevisiae Aga2 protein sequence (amino acid 1 to 87 of SEQ ID NO: 36), including its native 18 amino acid ER-target signal sequence (positions 1 to 18 of SEQ ID NO: 36); 2) Aga2 a spacer separate from the HA bulk (positions 88 and 89); 3) an influenza HA protein lacking the signal sequence (positions 90 to 600 of SEQ ID NO: 36) and lacking 36 C-terminal residues of HA, thereby removing it C-terminal membrane anchor and cytoplasmic tail; 4) triglycine spacer separating HA protein bulk from histidine label (positions 601-603 of SEQ ID NO: 36); and 5) C-terminal hexameric group Amino acid label (positions 604-609 of SEQ ID NO: 36). The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 36 is represented herein as SEQ ID NO:35. This fusion protein and the Tarmogen that express it can be called 75-15.

HA H1-Aga2 fusion protein (surface)

The fusion protein (shown as VK4), schematically shown in Figure 10B (upper right), was designed to express the influenza HA protein (driven by the TEF2 promoter) on the cell wall using the Aga2 sequence. In this construction, the protein is constructed such that the HA sequence is located at the N-terminus of the Aga2 sequence. This protein is localized to the outside of the cell wall of the yeast cell when it is expressed in a yeast which also exhibits native Aga1p (in this case, its natural promoter), and may also be present in the cell. A fusion protein comprising an influenza HA antigen (H1) is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID NO: 26): 1) Aga2 ER target signal sequence (amino acid 1 to 19 of SEQ ID NO: 26); 2) lack of signal sequence and C-terminal transmembrane domain (positions 20 to 533 of SEQ ID NO: 26) from A /PR/8/34 of H1 HA; 3) a spacer separating the HA protein bulk from the histidine label (positions 534 to 535 of SEQ ID NO: 26); 4) hexahistidine label (SEQ ID NO) :26 position 536 to 541); 5) enterokinase cleavage site (positions 542 to 548 of SEQ ID NO: 26); 6) Aga2 lacking the signal sequence (positions 549 to 614 of SEQ ID NO: 26). The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 26 is represented herein as SEQ ID NO: 25.

HA H5-Aga2 fusion protein (surface)

The fusion protein designated VK11 (similar to VK4 above, except that it includes the H5 HA protein from avian influenza (A/Vietnam/1203/04)) was designed to express the influenza HA H5 protein on the cell wall using the Aga2 sequence. In this construction, the protein is constructed such that the HA sequence is located at the N-terminus of the Aga2 sequence. This protein is localized to the outside of the cell wall of the yeast cell when it is expressed in the yeast, and may also be present in the cell. A fusion protein comprising an influenza HA antigen (H5) is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID NO: 22): 1) Aga2 ER target signal sequence (amino acid 1 to 19 of SEQ ID NO: 22); 2) lack of signal sequence and C-terminal transmembrane domain (positions 20 to 536 of SEQ ID NO: 22) from A /Vietnam/1203/04 H5 HA; 3) hexahistidine label (positions 537 to 542 of SEQ ID NO: 22); 4) enterokinase cleavage site (positions 543 to 548 of SEQ ID NO: 22) 5) Aga2 lacking the signal sequence (positions 549 to 616 of SEQ ID NO: 22). The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 22 is represented herein as SEQ ID NO:21.

HA H1-Cwp2 fusion protein (surface)

The fusion protein (shown as VK8) schematically shown in Figure 10B (bottom left) was designed to express the influenza HA protein on the cell wall (driven by the TEF2 promoter) using the Cwp2 sequence. This protein can be localized to the outside of the cell wall of the yeast cell and can also be present in the cell. A fusion protein comprising an influenza HA antigen (H1) is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID NO: 28): 1) Suc2 invertase signal sequence (amino acid 1 to 21 of SEQ ID NO: 28); 2) lack of signal sequence and C-terminal transmembrane domain (positions 22 to 535 of SEQ ID NO: 28) from A /PR/8/34 of H1 HA; 3) a spacer separating the HA protein bulk from the histidine label (positions 536 to 537 of SEQ ID NO: 28); 4) hexahistidine label (SEQ ID NO) Position: 538 to 543); 5) Enterokinase cleavage site (positions 544 to 549 of SEQ ID NO: 28); 6) Cwp2 lacking the signal sequence (positions 550 to 617 of SEQ ID NO: 28). The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 28 is represented herein as SEQ ID NO:27.

HA H5-Cwp2 fusion protein (surface)

The VK12 fusion protein (similar to VK8 above, except that it includes the H5 HA protein from avian influenza (A/Vietnam/1203/04)) was designed to express the influenza HA H5 protein on the cell wall using the Cwp2 sequence. This protein can be localized to the outside of the cell wall of the yeast cell and can also be present in the cell. A fusion protein comprising an influenza HA antigen (H5) is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID NO: 24): 1) Suc2 invertase signal sequence (amino acid 1 to 21 of SEQ ID NO: 24); 2) lack of signal sequence and C-terminal transmembrane domain (positions 22 to 536 of SEQ ID NO: 24) from A /Vietnam/1203/04 of H5 HA; 3) a spacer separating the HA protein bulk from the histidine label (positions 537 to 538 of SEQ ID NO: 24); 4) hexahistidine label (SEQ ID NO) : position 539 to 544); 5) enterokinase cleavage site (positions 545 to 550 of SEQ ID NO: 24); 6) Cwp2 lacking the signal sequence (positions 551 to 618 of SEQ ID NO: 24). The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 24 is represented herein as SEQ ID NO:23.

HA-fusion protein (surface) for protoplast spheroid expression

The fusion protein (shown as Lu002), schematically shown in Figure 10B (bottom right), was designed to represent influenza HA protein (driven by the TEF2 promoter) with a complete transmembrane domain on the plasma membrane of the yeast protoplast spheroid. This protein can be localized to the quality of the yeast protoplast spheroid The membrane may also be present in the cells.

Figure 11 depicts another Tarmogen that expresses a fusion protein (referred to above as VK8) that expresses influenza HA protein on the surface of yeast via cell wall protein 2 (cwp2). The histogram of HA on the surface of the yeast obtained by flow cytometry is shown in the lower right corner. These histograms show that this particular construct is a good indicator of HA on the cell surface compared to a single yeast vehicle (GI-1001 or YVEC).

Figures 12A-12G show histograms in which influenza HA proteins on the surface of a yeast vehicle have been expressed using various methods (described above and illustrated in Figure 10B). These experiments were performed under normal yeast culture conditions (i.e., without neutral pH conditions). Figures 12A-12C illustrate the performance of a Tarmogen expressing VK4 and TK75-15 fused in two different directions as described above using an Aga2 spacer or linker. Figure 12A shows the performance of a control (non-transformed) yeast (YEX). Figure 12B shows the performance of VK4 and Figure 12C shows the performance of TK75-15. Figures 12D-12G show other possible configurations for surface representation of HA. Figure 12D is again a yeast control (YEX). Figures 12E and 12F show the use of Cwp2 as a spacer arm for the performance of HA, a Tarmogen expressing VK8. These patterns also illustrate the effect of regulating the glycosylation of yeast on the expression of the protein. The Tarmogen (Fig. 12F), which exhibits deglycosylated VK8, can improve the surface performance of HA compared to the Tarmogen (Fig. 12E) which exhibits glycosylated VK8. Finally, Figure 12G shows the performance of HA by the Tarmogen expressing Lu002, a protoplast spheroid that expresses HA on the plasma membrane.

These results demonstrate that various antigens can be used on the surface (extracellular) of the yeast vehicle of the present invention.

Example 6

The following example describes the design of another influenza fusion protein yeast vaccine of the invention in which the combination of influenza proteins is expressed intracellularly by a yeast vehicle.

Tarmogen is designed to function as a matrix protein (M1) nuclear sheath protein (NP) and an ion channel protein extracellular sequence (M2e) as an intracellular fusion protein under the control of the TEF2 promoter (see Figure 13A, which illustrates the performance of this fusion protein and the performance of the second HA structure on the same plasmid). The M1 and NP (N1) sequences were obtained from the A/PR/8/34 influenza virus strain. 4xM2e represents two copies of the M2e sequence obtained from the A/PR/8/34 influenza virus strain and two copies of the M2e sequence obtained from the A/Viet Nam/1203/2004 influenza virus strain. The fusion protein comprising the M1-N1-4xM2e protein is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID NO: 16): 1) Sequence to prevent proteasome degradation (positions 1 to 6 of SEQ ID NO: 16) MADEAP (SEQ ID NO: 1); 2) influenza A/PR/8/34 M1 protein (position 7 of SEQ ID NO: 16) To 260); 3) a spacer separating the M1 protein from the NP protein (positions 261 to 262 of SEQ ID NO: 16); 4) influenza A/PR/8/34 NP protein (position 263 of SEQ ID NO: 16) To 760); 5) a spacer separating the NP protein from the M2e protein (positions 761 to 762 of SEQ ID NO: 16); 6) obtained from the influenza A/PR/8/34 M2 protein (SEQ ID NO: 16) First M2e (extracellular) protein at positions 763 to 787); 7) second M2e (extracellular) obtained from influenza A/Viet Nam/1203/2004 M2 protein (positions 788 to 811 of SEQ ID NO: 16) Protein; 8) a spacer separating the second M2e protein from the third M2e protein (positions 812 to 813 of SEQ ID NO: 16); 9) obtained from the influenza A/PR/8/34 M2 protein (SEQ ID NO: The third M2e (extracellular) protein at positions 16 to 838); 10) was obtained from influenza A/Viet Nam/1203/2004 M 2 protein (position 839 to 862 of SEQ ID NO: 16) (fourth M2e protein composed of (extracellular) protein; and 11) C-terminal hexahistidine label (positions 864 to 869 of SEQ ID NO: 16) ). The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 16 is represented herein as SEQ ID NO: 15.

Referring to Figures 13A and 13B, schematic representations of the use of the Tarmogen representing the M1-NP-4xM2e fusion protein (SEQ ID NO: 16) along with the associated additional structures are shown. Specifically, the fusion protein represented by SEQ ID NO: 16 can also be produced in a single plasmid which also contains a second structure encoding the HA protein under the control of the CUP1 promoter. The expression of this plasmid produces an intracellular representation of the M1-NP-4xM2e fusion protein and the HA protein. The performance of HA can also be Obtained using a second independent structure. Generation of other Tarmogens involves the use of the structure depicted in Figure 13B (corresponding to the VK8 structure described above) (which allows the HA protein to be expressed extracellularly (surface)). This fusion protein can be expressed in the same Tarmogen having the above M1-N1-4xM2e fusion protein alone or in combination with the HA structure represented by the above. A separate Tarmogen expressing a VK8 (surface HA) fusion protein, alone or in combination with an HA expressed internally, may also be provided in the vaccine together with the above-described Tarmogen expressing the M1-N1-4xM2e fusion protein. One of the advantages of providing a single fusion protein for both intracellular and extracellular expression is to ensure that sufficient antigen is available for B cell response and uptake of antigen presenting cells (such as dendritic cells) for cell-mediated immune responses. use.

Example 7

The following example describes the design of another influenza fusion protein yeast vaccine of the invention in which the combination of influenza proteins is expressed intracellularly by a yeast vehicle.

A Tarmogen expressing a NP-2xM2e fusion protein can be produced and a Tarmogen expressing a NP-4xM2e fusion protein can be produced.

The NP-4xM2e fusion protein was constructed in the same manner as the M1-N1-4xM2e fusion protein described in Example 6 and represented by SEQ ID NO: 16, except that the M1 portion of the structure was not included.

In the NP-2xM2e fusion protein, two copies of the NP (N1) and M2e sequences were obtained from the A/PR/8/34 influenza virus strain. A fusion protein comprising a N1-2xM2e protein is a single polypeptide having the following sequence components fused in the N-terminal to C-terminal framework (the amino acid sequence of the fusion protein is represented herein as SEQ ID NO: 18): 1) Sequence to prevent proteasome degradation (positions 1 to 6 of SEQ ID NO: 18) MADEAP (SEQ ID NO: 1); 2) influenza A/PR/8/34 NP protein (positions 7 to 503 of SEQ ID NO: 18) 3) a spacer separating the NP protein from the M2e protein (positions 504 to 505 of SEQ ID NO: 18); 4) obtained from the influenza A/PR/8/34 M2 protein (position 506 of SEQ ID NO: 18) a first M2e (extracellular) protein to 530); 5) a spacer separating the first M2e protein from the second M2e protein (position 531 of SEQ ID NO: 18); 6) a second M2e (extracellular) protein obtained from the influenza A/PR/8/34 M2 protein (positions 532 to 555 of SEQ ID NO: 18); and 7) followed by a C-terminal hexahistamine Triglycine spacers acid-labeled (positions 556 to 564 of SEQ ID NO: 18). The nucleic acid sequence encoding the fusion protein of SEQ ID NO: 18 is represented herein as SEQ ID NO: 17.

The fusion protein expressed in the yeast vehicle to produce the Tarmogen can be intracellularly expressed and can be combined with the performance of other structures (eg, HA-surface or HA-internal performance strategies as described in Example 6 above).

Example 8

The following example demonstrates that immunization with the Tarmogen, which expresses influenza HA as an extracellular protein, can elicit a humoral immune response.

In this example, a single dose of GI-8000-S, which is a fusion protein with Cwp2 (see Figure 10B, VK8) on the surface of yeast cells, is administered subcutaneously to mice, or at low doses. The live influenza virus is infected intravenously in mice. One week after immunization, the HI titer was measured. Another group of mice received 5YU of GI-8000-S. One week after immunization, the HI titer was measured. The results of this study are shown in Table 5. This data indicates that a single dose of the GI-8000-S vaccine is capable of eliciting a HA neutralizing antibody response without the addition of soluble purified proteins or the addition of a non-yeast based vaccine.

Example 9

Antibody production was initiated using Tarmogen, which displays various antigens studied. As discussed below, the invention disclosed herein provides methods of inducing humoral immune responses, including antibody responses.

GI-2001 (HIVAX-1), which produces an outer membrane protein of HIV-1 gp160 as a membrane protein in yeast cells, has been studied (Franzusoff et al., J. Biol. Chem. 270, 3154-3159 (1995) ). For complete lysates of intact yeast or spheroplasts, 2x10 ⁷ (2YU) of YVEC or HIVAX-1 strain (sponsored as live intact yeast cells or as procollagen globular) obtained weekly for three weeks The serum of mice administered in vivo was subjected to Western blot analysis. Western blotting methods demonstrate that mice produce antibodies against various yeast-derived proteins and the resulting antibody type depends on whether the mouse is injected with intact yeast or spheroplasts. Sera from mice immunized with HIVAX-1 protoplast spheroids instead of intact HIVAX-1 yeast or YVEC yeast or spheroplast spheroids appear to contain gp160-specific antibodies. Without being bound by theory, this finding may be due to gp160 being expressed as a membrane intrinsic protein in HIVAX-1 such that it is not exposed to B cells on the surface of intact yeast and is exposed to the outer surface of the spheroplast spheroid.

Serum obtained from mice vaccinated with OVAX (Tarmogen producing chicken ovalbumin) demonstrated the titer of anti-OVA antibody compared to the results obtained from HIVAX-1. This result appears to be due to the fact that chick ovalbumin is mainly secreted in the periplasmic space of the OVAX yeast strain and some soluble protein lines are released via the cell wall of the intact yeast. Thus, localization of a heterologous antigen within a recombinant yeast-based vaccine appears to determine whether antibodies are produced.

To further investigate the ability of OVAX yeast to induce anti-OVA antibodies, BALB/c mice were vaccinated with the antigens shown in the table below once a month. One month after the second vaccination, mice were inoculated with soluble chicken ovalbumin (without adjuvant). Mice that mimicked vaccinated PBS did not establish an anti-OVA antibody response after a single challenge with soluble OVA on day 56. In contrast, two vaccinated 2 x 10 ⁷ (2YU) OVAX yeast inoculated mice established a high titer anti-OVA antibody response following challenge with soluble OVA (without adjuvant). In mice immunized with OVAX, followed by soluble OVA (examples based on non-yeast vaccines) or co-administered with OVAX and OVA-Alum, and then stimulated with soluble OVA (especially in single dose OVAX plus OVA) High-cost anti-OVA antibodies were observed after -Alum, and this study demonstrated antigen-saving effects. Figures 14A and 14B also illustrate the results of similar experiments.

Referring to Figures 14A and 14B, the results of T cell initiation (Figure 14B) and antibody production (Figure 14A) of the three protocols used are shown. In Figure 14A, protocol A (control), in which only PBS was administered, showed no ovalbumin-specific antibody titers were detected when challenged with PBS. In protocol B, PBS was primed on days 0 and 28, and when challenged with soluble ovalbumin (ova) on day 56, ovalbumin-specific antibody titers were not detected until day 65. In protocol C, a yeast vehicle expressing ovalbumin (OVAX) was used, and when stimulated with soluble ovalbumin, rapid production of high antibody titers was observed. Figure 14B shows the results of using various amounts of soluble ovalbumin for in vitro re-excitation of T cell activation assays obtained from T cells of mice immunized with each of the above three protocols. Scheme C is effective in inducing a cell-mediated immune response.

Example 10

The following example demonstrates that Tarmogen producing Gag can elicit antigen-specific helper T cells.

The purpose of the following examples is to determine the ability of GI-2010 (Tarmogen that produces HIV-1 Gag protein such as cytosolic protein) to elicit antigen-specific helper T cells for antibody production. Each group of 5 BALB/c mice was injected with normal saline, 2YU YVEC or 2YU GI-2010 (subcutaneous injection on days 0, 7 and 21) or 1 x 10 ⁷ pfu MVA-control or 1 × 10 ⁷ MVA- UGD (recombinant modified vaccinia vaccine Ankara virus encoding HIV-1 Gag; intraperitoneal injection on day 0 and day 21). Subsequently, mice immunized with physiological saline or yeast were injected with 5 μg of recombinant p24 Gag protein in physiological saline (without adjuvant) on the 28th day. Anti-p24 Gag antibody titers of mouse serum samples isolated throughout the study were determined by ELISA. The results shown in Figure 15 demonstrate that mice immunized with GI-2010 did not produce detectable p24 Gag specific antibodies unless they were stimulated by soluble Gag protein. Since mice injected with normal saline or YVEC did not produce antibodies, this data indicates that GI-2010 triggers helper T cells in the vaccinated mice to stimulate B cell responses induced by soluble Gag protein (without adjuvant). .

This example demonstrates that Tarmogen can induce helper T cells for antibody production and that antigen localization within the yeast plays an important role in the amount of antibody produced. For example, studies have shown that yeasts that display HBsAg as a yeast cell surface protein can induce anti-HBsAg antibodies. See, for example, M. P. Schreuder et al, Vaccine, 14(5): 383-8 (1996).

Example 11

The following examples demonstrate that the yeast-based vaccine of the present invention has an adjuvant effect when mixed with a non-yeast based antigen preparation.

In this experiment, GI-8002 (Tarmogen producing A/PR/8/34 HA such as cytosolic protein) (see Example 2) was used. In summary, BALB/c mice (5 per group) were subcutaneously immunized with 5YU (5 × 10 ⁷ ) GI-8002 yeast cells on day 1 and day 22, and 1 μg or 10 μg of BPL-inoculated alone. Live influenza virus (A/PR/8/34), or immunization with 5YU GI-8002 plus a specified amount of BPL-inactivated influenza virus. Four weeks after the second dose, serum was collected and a HI assay was performed to determine the presence of neutralizing antibodies. The results of this experiment demonstrate that yeast can have an adjuvant effect when mixed only with BPL-inactivated virus to induce neutralizing antibodies.

The disclosures of all of the patents, patent applications, and publications cited herein are hereby incorporated by reference in its entirety herein in its entirety in the extent the the the the the

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While the embodiments of the present invention have been described in detail, it is obvious that those skilled in the art can make modifications and adjustments to the embodiments. However, it should be expressly understood that such modifications and adaptations are within the scope of the invention as described in the following claims.

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<210> 6

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<212> PRT

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<223> Synthesis

<400> 6

<210> 7

<211> 240

<212> PRT

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<220>

<223> Synthesis

<400> 7

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<211> 750

<212> DNA

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<223> Synthesis

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<212> PRT

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<223> Synthesis

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<212> PRT

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<220>

<223> Synthesis

<400> 11

<210> 12

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<212> PRT

<213> Influenza A virus

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<223> Synthesis

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<210> 14

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<212> DNA

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<223> Synthesis

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<211> 564

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<223> Synthesis

<400> 18

<210> 19

<211> 1884

<212> DNA

<213> Labor

<220>

<223> Synthesis

<400> 19

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<212> PRT

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<220>

<223> Synthesis

<400> 20

<210> 21

<211> 1851

<212> DNA

<213> Labor

<220>

<223> Synthesis

<400> 21

<210> 22

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<212> PRT

<213> Labor

<220>

<223> Synthesis

<400> 22

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<212> DNA

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<220>

<223> Synthesis

<400> 23

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<223> Synthesis

<400> 24

<210> 25

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<212> DNA

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<220>

<223> Synthesis

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<223> Synthesis

<400> 26

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<212> DNA

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<223> Synthesis

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<223> Synthesis

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<213> Influenza virus

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<212> DNA

<213> Influenza virus

<400> 31

<210> 32

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<212> PRT

<213> Influenza virus

<400> 32

<210> 33

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<212> DNA

<213> Influenza virus

<400> 33

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<213> Influenza virus

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Claims (74)

A vaccine comprising: a) a first yeast vehicle comprising at least one heterologous intracellular antigen; and b) a second yeast vehicle comprising at least one heterologous extracellular antigen. A vaccine comprising: a) a first yeast vehicle comprising at least one heterologous intracellular antigen and at least one heterologous extracellular antigen; and b) comprising at least one heterologous intracellular antigen or at least one heterologous extracellular A second yeast agent for the antigen. A vaccine comprising: a) a yeast vehicle comprising at least one heterologous intracellular antigen and at least one heterologous extracellular antigen; and b) a non-yeast based composition comprising at least one (a An antigen contained in a yeast vehicle or an antigen obtained from the same pathogen or disease, wherein the non-yeast based composition is selected from the group consisting of a DNA vaccine, a protein subunit vaccine, and a dead or inactivated pathogen. group. The vaccine of claim 3, wherein the yeast vehicle of (a) is formulated to be delivered by a different route of administration than the non-yeast based composition of (b). The vaccine according to any one of claims 1 to 3, wherein the intracellular antigen is an antigen expressed internally by a pathogen. The vaccine of any one of claims 1 to 3, wherein the extracellular antigen is a structurally conserved antigen of the same type of pathogen. The vaccine of any one of claims 1 to 3, wherein the extracellular antigen is an antigen that is expressed on the surface of the pathogen. The vaccine of any one of claims 1 to 3, wherein the extracellular antigen is the same type of disease A structurally variable antigen in a protoplast. The vaccine of any one of claims 1 to 3, wherein the antigen is from an infectious pathogen. A vaccine comprising: a) a yeast vehicle; and b) an influenza virus fusion protein expressed by the yeast agent, the influenza virus fusion protein comprising a protein selected from the group consisting of influenza matrix (M1) and influenza ion channel protein (M2) At least a portion of the constituent influenza proteins. A vaccine comprising: a) a first yeast vehicle expressing an influenza virus fusion protein comprising a protein selected from the group consisting of influenza matrix (M1) protein, influenza ion channel protein (M2), and nuclear sheath (NP) protein At least a portion of the constituent influenza proteins; and b) at least one other yeast agent that expresses an influenza virus fusion protein comprising a protein selected from the group consisting of a hemagglutinin (HA) protein and a neuraminidase ( NA) At least a portion of the influenza protein of the group of proteins. A vaccine comprising: a) a yeast vehicle; and b) an influenza virus fusion protein expressed by the yeast vehicle, the influenza virus fusion protein comprising: at least one selected from the group consisting of influenza matrix (M1) proteins, influenza ion channels At least a portion of a first influenza protein of a population consisting of a protein (M2) and a nuclear sheath (NP) protein; and at least one selected from the group consisting of a hemagglutinin (HA) protein and a neuraminidase (NA) protein At least a portion of the second influenza protein. A vaccine comprising: a) a yeast vehicle; b) a first influenza virus fusion protein expressed by the yeast agent, the first influenza virus fusion protein comprising at least one selected from the group consisting of influenza matrix (M1) protein, influenza from At least a portion of an influenza protein comprising a population of subchannel protein (M2) and a nuclear sheath (NP) protein; and c) a second influenza virus fusion protein expressed by the yeast vehicle, the second influenza virus fusion protein comprising at least At least a portion of an influenza protein selected from the group consisting of a hemagglutinin (HA) protein and a neuraminidase (NA) protein. A vaccine comprising: a) a yeast vehicle; and b) at least one first influenza virus protein selected from the group consisting of influenza matrix (M1) protein, influenza ion channel protein (M2), and nuclear sheath (NP) protein At least a portion; and c) at least one portion of a second influenza protein selected from the group consisting of a hemagglutinin (HA) protein and a neuraminidase (NA) protein. The vaccine of any one of claims 10 to 14, wherein the HA protein is selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and A group of H16. The vaccine of any one of claims 10 to 14, wherein the HA protein is selected from the group consisting of H1, H2 and H3. The vaccine of any one of claims 10 to 14, wherein the HA protein is H5. The vaccine of any one of claims 10 to 14, wherein the NA protein is selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8 and N9. The vaccine of any one of claims 10 to 14, wherein the NA protein is selected from the group consisting of N1 and N2. The vaccine of any one of claims 9 to 14, wherein the M1 protein is intracellular to the yeast vehicle. The vaccine of any one of claims 9 to 14, wherein the M2 protein is intracellular, extracellular, or intracellular and extracellular to the yeast vehicle. The vaccine of any one of claims 9 to 14, wherein the M2 protein is M2e. The vaccine of any one of claims 9 to 14, wherein the NP protein is intracellular to the yeast vehicle. The vaccine of any one of claims 10 to 14, wherein the HA protein or the NA protein is extracellular to the yeast vehicle. The vaccine of claim 24, wherein the HA protein or the NA protein is also intracellular to the yeast vehicle. The vaccine according to any one of claims 9 to 14, wherein the influenza virus fusion protein comprises an amino acid sequence represented by SEQ ID NO: 1 (MADEAP) at its N-terminus. The vaccine of any one of claims 9 to 14, wherein the influenza virus fusion protein comprises at its N-terminus or C-terminus at least a portion of an Aga2 protein sufficient to target the fusion protein to the cell wall of the yeast vehicle. The vaccine of any one of claims 9 to 14, wherein the influenza virus fusion protein comprises at its N-terminus or C-terminus at least a portion of a Cwp2 protein sufficient to target the fusion protein to the cell wall of the yeast vehicle. A vaccine comprising: a) a yeast vehicle; and b) an influenza virus fusion protein comprising an M1 antigen represented by the yeast vehicle as a single intracellular fusion protein, the fusion protein consisting of SEQ ID NO:4 . A vaccine comprising: a) a yeast vehicle; and b) an influenza virus fusion protein comprising an H1 antigen represented by the yeast vehicle as a single extracellular fusion protein, the fusion protein being selected from the group consisting of SEQ ID NO The composition of the amino acid sequence of the group consisting of: 6 and SEQ ID NO: 20. A vaccine comprising: a) a yeast vehicle; and b) an influenza virus fusion protein comprising a single cell represented by the yeast vehicle The H1 antigen of the outer fusion protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 36. A vaccine comprising: a) a yeast vehicle; and b) an influenza virus fusion protein comprising an H5 antigen represented by the yeast vehicle as a single extracellular fusion protein, the fusion protein being selected from the group consisting of SEQ ID NO The composition of the amino acid sequence of the group consisting of: SEQ ID NO: 22 and SEQ ID NO: 24. A vaccine comprising: a) a yeast vehicle; and b) an influenza virus fusion protein comprising an M1 antigen, an N1 antigen, and four M2e antigens, the yeast agent being expressed as a single intracellular fusion protein, the fusion The protein consists of SEQ ID NO:16. A vaccine comprising: a) a yeast vehicle; and b) an influenza virus fusion protein comprising an N1 antigen and two M2e antigens, the yeast agent being expressed as a single intracellular fusion protein, the fusion protein being SEQ. ID NO: 18 composition. A vaccine comprising: a) a yeast vehicle; and b) an influenza virus fusion protein comprising an H3 antigen and an N2 antigen, the yeast agent being expressed as a single extracellular fusion protein. The vaccine of any one of claims 1-3, 10-14 or 29-35, wherein the expression of the fusion protein is under the control of an inducible promoter. The vaccine of any one of claims 1-3, 10-14, or 29-35, wherein the promoter is selected from the group consisting of CUP1 and TEF2 . The vaccine of any one of claims 1-3, 10-14 or 29-35, wherein the yeast vehicle is selected from the group consisting of whole yeast, yeast protoplast spheroid, yeast cytoplasm, yeast residue a yeast cell wall preparation and a subcellular yeast membrane extract or a fraction thereof. The vaccine of any one of claims 1-3, 10-14 or 29-35, wherein the yeast cell or yeast protoplast globular system for preparing the yeast vehicle is subjected to a recombinant nucleic acid encoding the fusion protein The molecular transformation is such that the fusion protein is recombined by the yeast cell or the yeast protoplast spheroid. The vaccine of any one of claims 1-3, 10-14 or 29-35, wherein the yeast cell or yeast protoplast globular system expressing the fusion protein is used to produce a yeast cytoplasmic body, yeast A yeast vehicle, a yeast cell wall preparation or a subcellular yeast membrane extract or a portion thereof. The vaccine of any one of claims 1-3, 10-14 or 29-35, wherein the yeast vehicle is derived from a non-pathogenic yeast. The vaccine of any one of claims 1-3, 10-14 or 29-35, wherein the yeast vehicle is derived from a yeast selected from the group consisting of Saccharomyces , Schizosaccharomyces , A group consisting of Kluveromyces , Hansenula , Candida , and Pichia . The vaccine of any one of claims 1-3, 10-14 or 29-35, wherein the genus Saccharomyces is S. cerevisiae . The vaccine of any one of claims 1-3, 10-14, or 29-35, wherein the vaccine further comprises dendritic cells, wherein the dendritic cells have been loaded with the yeast vehicle in the cells. The vaccine of any one of claims 1-3, 10-14 or 29-35, further comprising at least one biological response modifier. A vaccine according to any one of claims 1-3, 10-14 or 29-35 for use in the preparation of a formulation for inducing an antigen-specific immune response. A vaccine according to any one of claims 1-3, 10-14 or 29-35 for use in the preparation of a formulation for the treatment or prevention of a disease or condition. The use of claim 47, wherein the disease or condition is a pathogen infection. A vaccine according to any one of claims 10-14 or 29-35 for use in the preparation of a formulation for eliciting an antigen-specific cell-mediated immune response against influenza antigen. A vaccine according to any one of claims 10-14 or 29-35 for use in the preparation of a formulation for protecting an animal against influenza infection. A vaccine according to any one of claims 10-14 or 29-35 for use in the preparation of a formulation for immunizing a population of individuals at risk of contracting influenza. A vaccine according to any one of claims 10-14 or 29-35 for use in the preparation of a formulation for treating a population of individuals infected with influenza. A yeast vehicle, at least one heterologous intracellular antigen, and at least one heterologous extracellular antigen are used to prepare a formulation for inducing a cell-mediated immune response and a humoral immune response. The use of claim 53, wherein the yeast vehicle is formulated for parenteral delivery or intranasal delivery. The use of claim 53, wherein the intracellular antigen is an antigen expressed internally by the pathogen. The use of claim 53, wherein the extracellular antigen is a structurally conserved antigen of the same type of pathogen. The use of claim 53, wherein the extracellular antigen is an antigen that is expressed on the surface of the pathogen. The use of claim 53, wherein the extracellular antigen is in the same type of pathogen Construct a variable antigen. A use of a first composition comprising a yeast vehicle in combination with a second composition for the preparation of a formulation for inducing a cell-mediated immune response and a humoral immune response in an individual, the yeast vehicle comprising at least one different a source intracellular antigen and at least one heterologous extracellular antigen, the second composition comprising: a) a yeast vehicle comprising at least one of the heterologous antigens for use in the first composition or obtained from the same pathogen Or an antigen of a disease, wherein the antigen is extracellular or intracellular and extracellular to the yeast; b) a yeast membrane or cell wall particle containing a heterologous source for use in the first composition At least one of the antigens or an antigen obtained from the same pathogen or disease; c) a yeast vehicle mixed with at least one of the heterologous antigens used in the first composition or an antigen obtained from the same pathogen or disease; d) a DNA vaccine encoding at least one of the antigens used in the first composition or an antigen obtained from the same pathogen or disease; e) a protein subunit vaccine comprising the antigens for use in the first composition In the middle At least one or an antigen obtained from the same pathogen or disease or an antigen obtained from the same pathogen or disease; or f) a dead or inactivated pathogen comprising at least one of the heterologous antigens for use in the first composition. A method of preparing a yeast vaccine according to any one of claims 1-3, 10-14 or 32-38, which comprises culturing a yeast vehicle comprising the antigen at a pH greater than pH 5.5. A method for preparing an influenza vaccine, comprising transfecting a yeast vehicle with an influenza antigen fusion protein, wherein the influenza antigen fusion protein comprises: selected from the group consisting of influenza matrix (M1) protein, influenza ion channel protein (M2), and influenza virus nuclear sheath At least a portion of an influenza virus protein of a group consisting of (NP) proteins; and an egg selected from hemagglutinin (HA) At least a portion of the influenza protein of the group consisting of the white and neuraminidase (NA) proteins. The method of claim 61, comprising culturing the yeast vehicle at a pH greater than pH 5.5. The method of claim 61, further comprising loading the yeast vehicle with an influenza virus protein. The method of claim 61, further comprising mixing the influenza virus antigen with the yeast vehicle. The method of claim 61, further comprising the influenza virus antigen physically attached to the yeast vehicle. The method of claim 61, further comprising formulating the transformed yeast vehicle for injection into the individual. The method of claim 61, further comprising formulating the transformed yeast vehicle for intranasal administration to the individual. A kit for preparing a formulation for inducing a cell-mediated immune response, a humoral immune response, or a combination thereof in an individual, the kit comprising: a plurality of yeast vehicles, wherein each of the yeast vehicles comprises at least one intracellular a heterologous antigen or at least one extracellular heterologous antigen; and instructions for using the yeast vehicle to prepare the formulation. The kit of claim 68, wherein the yeast vectors recombine the antigens. The kit of claim 68, further comprising at least one other composition selected from the group consisting of: a) a yeast membrane or cell wall particle containing at least one of the heterologous antigens or obtained from the same An antigen of a pathogen or disease; b) a yeast vehicle, and at least one of the heterologous antigens or obtained from the same pathogen Or a mixture of antigens of the disease; c) a DNA vaccine encoding at least one of the antigens or an antigen obtained from the same pathogen or disease; d) a protein subunit vaccine comprising at least one of the antigens or obtained from An antigen of the same pathogen or disease or an antigen obtained from the same pathogen or disease; and e) a dead or inactivated pathogen comprising at least one of the heterologous antigens. A kit of claim 68, wherein the intracellular antigen is an antigen that is manifested internally by the pathogen. A kit of claim 68, wherein the extracellular antigen is a structurally conserved antigen of the same type of pathogen. A kit of claim 68, wherein the extracellular antigen is an antigen that is expressed on the surface of the pathogen. A kit of claim 68, wherein the extracellular antigen is a structurally variable antigen of the same type of pathogen.